Methods of producing rpe cells and compositions of rpe cells

ABSTRACT

The present invention provides improved methods for producing RPE cells from human embryonic stem cells or from other human pluripotent stem cells. The invention also relates to human retinal pigmented epithelial cells derived from human embryonic stem cells or other human multipotent or pluripotent stem cells. hRPE cells derived from embryonic stem cells am molecularly distinct from adult and fetal-derived RPE cells, and are also distinct from embryonic stem cells. The hRPE cells described hemin are useful for treating retinal degenerative diseases.

RELATED APPLICATIONS

This application claims the benefit of priority to U.S. provisionalapplication Nos. 60/998,766, filed Oct. 12, 2007, 60/998,668, filed Oct.12, 2007, 61/009,908, filed Jan. 2, 2008, and 61/009,911, filed Jan. 2,2008. The disclosures of each of the foregoing applications are herebyincorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

The retinal pigment epithelium (RPE) is the pigmented cell layer justoutside the neurosensory retina. This layer of cells nourishes retinalvisual cells, and is attached to the underlying choroid (the layer ofblood vessels behind the retina) and overlying retinal visual cells. TheRPE acts as a filter to determine what nutrients reach the retina fromthe choroid. Additionally, the RPE provides insulation between theretina and the choroid. Breakdown of the RPE interferes with themetabolism of the retina, causing thinning of the retina. Thinning ofthe retina can have serious consequences. For example, thinning of theretina may cause “dry” macular degeneration and may also lead to theinappropriate blood vessel formation that can cause “wet” maculardegeneration).

Given the importance of the RPE in maintaining visual and retinalhealth, there have been significant efforts in studying the RPE and indeveloping methodologies for producing RPE cells in vitro. RPE cellsproduced in vitro could be used to study the developments of the RPE, toidentify factors that cause the RPE to breakdown, or to identify agentsthat can be used to stimulate repair of endogenous RPE cells.Additionally, RPE cells produced in vitro could themselves be used as atherapy for replacing or restoring all or a portion of a patient'sdamaged RPE cells. When used in this manner, RPE cells may provide anapproach to treat macular degeneration, as well as other diseases andconditions caused, in whole or in part, by damage to the RPE.

The use of RPE cells produced in vitro for screening or as a therapeuticrelies on methods that can be used to produce large numbers of RPE cellsin a systematic, directed manner. Such systematized differentiationmethods would provide significant advantages over previous schemes basedon, for example, spontaneous differentiation of RPE cells fromtransformed cell lines or other sources.

SUMMARY OF THE INVENTION

The present invention provides a method for differentiating RPE cellsfrom human pluripotent stem cells, such as human embryonic stem cellsand human induced pluripotent stem cells. The method is used to producelarge numbers of differentiated RPE cells for use in screening assays,to study the basic biology of the RPE, and as therapeutics. As describedherein, RPE cells differentiated from pluripotent stem cells, such ashuman embryonic stem cells, using this approach are molecularly distinctfrom human embryonic stem cells, as well as from adult and fetal-derivedRPE cells.

The present invention also provides preparations and pharmaceuticalpreparations of RPE cells derived from human pluripotent stem cells.Such RPE cell preparations are molecularly distinct from human embryonicstem cells, as well as from adult and fetal-derived RPE cells.

The present invention provides, for the first time, a detailed molecularcharacterization of RPE cells differentiated from human embryonic stemcells. The detailed characterization includes comparisons to RPE cellsderived from other sources (e.g., adult RPE cells and fetal RPE cells),as well as to human embryonic stem cells. This analysis not onlyprovides a deeper understanding of RPE cells, but it also revealed thatRPE cells differentiated from human embryonic stem cells have distinctmolecular properties that distinguish these cells from previouslydescribed RPE cells.

The present invention provides preparations of RPE cells, includingsubstantially purified preparations of RPE cells. Exemplary RPE cellsare differentiated from human pluripotent stem cells, such as humanembryonic stem cells or iPS cells. Human pluripotent stem cell-derivedRPE cells can be formulated and used to treat retinal degenerativediseases. Additionally, human pluripotent stem cell-derived RPE cellscan be used in screening assays to identify agents that modulate RPEcell survival (in vitro and/or in vivo), to study RPE cell maturation,or to identify agents that modulate RPE cell maturation. Agentsidentified using such screening assays may be used in vitro or in vivoand may provide additional therapeutics that can be used alone or incombination with RPE cells to treat retinal degenerative diseases.

The present invention provides improved methods for the production ofRPE cells from embryonic stem cells or other pluripotent stem cells. Themethods of the invention can be used to produce differentiated RPEcells. Optionally, the level of maturation, as assessed by pigmentationlevels, of the differentiated RPE cells can be modulated so thatdifferentiated RPE cells, mature RPE cells, or mixtures thereof areproduced. Also provided are improved methods for the treatment of eyedisorders. In particular, these methods involve the use of RPE cellsderived from human embryonic stem cells to treat or ameliorate thesymptoms of eye disorders, particularly eye disorders caused orexacerbated, in whole or in part, by damage to or breakdown of theendogenous RPE layer.

In certain aspects, the invention provides a method for producing aculture of retinal pigment epithelial (RPE) cells. In certainembodiments, the culture is a substantially purified culture containingat least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or greater than 99% differentiated RPE cells (at least 75% of theculture is a differentiated RPE cell, regardless of level of maturity).In certain embodiments, the substantially purified culture contains atleast 30%, 35%, 40% or 45% mature differentiated RPE cells. In certainembodiments, the substantially purified culture contains at least 50%mature differentiated RPE cells. In other embodiments, the substantiallypurified culture contains at least 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99%mature differentiated RPE cells. In certain embodiments, thedifferentiated RPE cells are derived from human embryonic stem cells,human iPS cells, or other pluripotent stem cells.

In certain embodiments, the method comprising the steps of

a) providing human embryonic stem cells;

b) culturing the human embryonic stem cells as embryoid bodies innutrient rich, low protein medium, which medium optionally containsserum free B-27 supplement;

c) culturing the embryoid bodies as an adherent culture in nutrientrich, low protein medium, which medium optionally contains serum freeB-27 supplement;

d) culturing the adherent culture of cells of (c) in nutrient rich, lowprotein medium, which medium does not contain serum free B-27supplement;

e) culturing the cells of (d) in medium capable of supporting growth ofhigh-density somatic cell culture, whereby RPE cells appear in theculture of cells.

f) contacting the culture of (e) with an enzyme;

g) selecting the RPE cells from the culture and transferring the RPEcells to a separate culture containing medium supplemented with a growthfactor to produce an enriched culture of RPE cells; and

h) propagating the enriched culture of RPE cells to produce asubstantially purified culture of RPE cells.

In certain other aspects, the invention provides a method of producing amature retinal pigment epithelial (RPE) cell, said method comprising thesteps of

a) providing human embryonic stem cells;

b) culturing the human embryonic stem cells as embryoid bodies innutrient rich, low protein medium, which medium optionally containsserum free B-27 supplement;

c) culturing the embryoid bodies as an adherent culture in nutrientrich, low protein medium, which medium optionally contains serum freeB-27 supplement;

d) culturing the adherent culture of cells of step (c) in nutrient rich,low protein medium, which medium does not contain serum free B-27supplement;

e) culturing the cells of (d) in medium capable of supporting growth ofhigh-density somatic cell culture, whereby RPE cells appear in theculture of cells

f) contacting the culture of (e) with an enzyme;

g) selecting the RPE cells from the culture and transferring the RPEcells to a separate culture containing medium supplemented with a growthfactor to produce an enriched culture of RPE cells;

h) propagating the enriched culture of RPE cells; and

i) culturing the enriched culture of RPE cells to produce mature RPEcells.

In certain embodiments of any of the foregoing, the substantiallypurified culture of RPE cells may contain both differentiated RPE cellsand mature differentiated RPE cells. Amongst the mature RPE cells, thelevel of pigment may vary. However, the mature RPE cells can bedistinguished visually from the RPE cells based on the increased levelof pigmentation and the more columnar shape.

In certain embodiments, the percentage of mature differentiated RPEcells in the culture can be reduced by decreasing the density of theculture. Thus, in certain embodiments, the method further comprisessubculturing a population of mature RPE cells to produce a culturecontaining a smaller percentage of mature RPE cells.

In certain embodiments, the medium used when culturing the cells asembryoid bodies may be selected from any medium appropriate forculturing cells as embryoid bodies. For example, one of skill in the artcan select amongst commercially available or proprietary media. Anymedium that is capable of supporting high-density cultures may be used,such as medium for viral, bacterial, or eukaryotic cell culture. Forexample, the medium may be high nutrient, protein-free medium or highnutrient, low protein medium. For example, the human embryonic stemcells may be cultured in MDBK-GM, OptiPro SFM, VP-SFM, EGM-2, orMDBK-MM. In certain embodiments the medium may also contain B-27supplement.

In certain embodiments, the medium described herein may also besupplemented with one or more growth factors. Growth factors that may beused include, for example, EGF, bFGF, VEGF, and recombinant insulin-likegrowth factor. The medium may also contain supplements such as heparin,hydrocortisone, ascorbic acid, serum (such as, for example, fetal bovineserum), or a growth matrix (such as, for example, extracellular matrixfrom bovine corneal epithelium, matrigel (BD biosciences), or gelatin).

In certain embodiments, mechanical or enzymatic methods are used toselect RPE cells from amongst clusters of non-RPE cells in a culture ofembryoid body, or to facilitate sub-culture of adherent cells. Exemplarymechanical methods include, but are not limited to, tituration with apipette or cutting with a pulled needle. Exemplary enzymatic methodsinclude, but are not limited to, any enzymes appropriate fordisassociating cells (e.g., trypsin, collagenase, dispase). In certainembodiments, a non-enzymatic solution is used to disassociate the cells,such as a high EDTA-containing solution such as, for example,Hanks-based cell disassociation buffer.

In certain embodiments, for any of the above articulated steps, thecells are cultured for between about 3 days and 45 days, such as 7 days,7-10 days, 7-14 days, or 14-21 days.

In certain embodiments the cells are cultured for about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, or about 46 days. In certain embodiments, the cells arecultured for less than or equal to about: 45, 40, 35, 30, 25, 21, 20,18, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 days. Notethat, for each of the above articulated method steps, the cells may becultured for the same period of time at each step or for differingperiods of time at one or more of the steps.

In certain embodiments, the RPE cells are further cultured to produce aculture of mature RPE cells. Both RPE cells and mature RPE cells aredifferentiated RPE cells. However, mature RPE cells are characterized byincreased level of pigment in comparison to differentiated RPE cells.The level of maturity and pigmentation can be modulated by increasing ordecreasing the density of the culture of differentiated RPE cells. Thus,a culture of RPE cells can be further cultured to produce mature RPEcells. Alternatively, the density of a culture containing mature RPEcells can be decreased to decrease the percentage of maturedifferentiated RPE cells and increase the percentage of differentiatedRPE cells.

The medium used to culture the RPE cells is any medium appropriate forcell culture, and can be selected by the skilled person. For example,any medium that is capable of supporting high-density cultures may beused, such as medium for viral, bacterial, or animal cell culture. Forexample, the cells described herein may be cultured in VP-SFM, EGM-2,and MDBK-MM.

In certain embodiments of any of the foregoing, said substantiallypurified culture of RPE cells (with or without mature RPE cells) arefrozen for storage. The cells may be stored by any appropriate methodknown in the art, e.g., cryogenically frozen and may be frozen at anytemperature appropriate for storage of the cells. For example, the cellsmay be frozen at approximately −20° C., −80° C., −120° C., or at anyother temperature appropriate for storage of cells. Cryogenically frozencells are stored in appropriate containers and prepared for storage toreduce risk of cell damage and maximize the likelihood that the cellswill survive thawing. In other embodiments, RPE cells are maintained atroom temperature, or refrigerated at, for example, approximately 4° C.

In certain embodiments of any of the foregoing, the method is performedin accordance with Good Manufacturing Practices (GMP). In certainembodiments of any of the foregoing, the human embryonic stem cells fromwhich the RPE cells are differentiated were derived in accordance withGood Manufacturing Practices (GMP). In certain embodiments of any of theforegoing, the human embryonic stem cells from which the RPE cells aredifferentiated were derived from one or more blastomeres removed from anearly stage embryo without destroying the remaining embryo.

In certain embodiments of any of the foregoing, the method is used toproduce a preparation comprising at least 1×10⁵ RPE cells, at least5×10⁵ RPE cells, at least 1×10⁶ RPE cells, at least 5×10⁶ RPE cells, atleast 1×10⁷ RPE cells, at least 2×10⁷ RPE cells, at least 3×10⁷ RPEcells, at least 4×10⁷ RPE cells, at least 5×10⁷ RPE cells, at least6×10⁷ RPE cells, at least 7×10⁷ RPE cells, at least 8×10⁷ RPE cells, atleast 9×10⁷ RPE cells, at least 1×10⁸ RPE cells, at least 2×10⁸ RPEcells, at least 5×10⁸ RPE cells, at least 7×10 RPE cells, or at least1×10⁹ RPE cells. In certain embodiments, the number of RPE cells in thepreparation includes differentiated RPE cells, regardless of level ofmaturity and regardless of the relative percentages of differentiatedRPE cells and mature RPE cells. In other embodiments, the number of RPEcells in the preparation refers to the number of either differentiatedRPE cells or mature RPE cells.

In certain embodiments, the method further comprises formulating thedifferentiated RPE cells and/or differentiated mature RPE cells toproduce a preparation of RPE cells suitable for transplantation.

In another aspect, the invention provides a method for treating orpreventing a condition characterized by retinal degeneration, comprisingadministering to a subject in need thereof an effective amount of apreparation comprising RPE cells, which RPE cells are derived from humanembryonic stem cells, iPS cells, or other pluripotent stem cells.Conditions characterized by retinal degeneration include, for example,Stargardt's macular dystrophy, age related macular degeneration (dry orwet), diabetic retinopathy, and retinitis pigmentosa. In certainembodiments, the RPE cells are derived from human pluripotent stem cellsusing one or more of the methods described herein.

In certain embodiments, the preparation was previously cryopreserved andwas thawed before transplantation.

In certain embodiments, the method of treating further comprisesadministration of cyclosporin or one or more other immunosuppressants.When immunosuppressants are used, they may be administered systemicallyor locally, and they may be administered prior to, concomitantly with,or following administration of the RPE cells. In certain embodiments,immunosuppressive therapy continues for weeks, months, years, orindefinitely following administration of RPE cells.

In certain embodiments, the method of treatment comprises administrationof a single dose of RPE cells. In other embodiments, the method oftreatment comprises a course of therapy where RPE cells are administeredmultiple times over some period. Exemplary courses of treatment maycomprise weekly, biweekly, monthly, quarterly, biannually, or yearlytreatments. Alternatively, treatment may proceed in phases wherebymultiple doses are required initially (e.g., daily doses for the firstweek), and subsequently fewer and less frequent doses are needed.Numerous treatment regimens are contemplated.

In certain embodiments, the administered RPE cells comprise a mixedpopulation of differentiated RPE cells and mature RPE cells. In otherembodiments, the administered RPE cells comprise a substantiallypurified population of either differentiated RPE cells or mature RPEcells. For example, the administered RPE cells may contain less than25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1% ofthe other RPE cell-type.

In certain embodiments, the RPE cells are formulated in apharmaceutically acceptable carrier or excipient.

In certain embodiments, the preparation comprising RPE cells istransplanted in a suspension, matrix or substrate. In certainembodiments, the preparation is administered by injection into thesubretinal space of the eye. In certain embodiments, about 10⁴ to about10⁶ cells are administered to the subject. In certain embodiments, themethod further comprises the step of monitoring the efficacy oftreatment or prevention by measuring electroretinogram responses,optomotor acuity threshold, or luminance threshold in the subject. Themethod may also comprise monitoring the efficacy of treatment orprevention by monitoring immunogenicity of the cells or migration of thecells in the eye.

In certain aspects, the invention provides a pharmaceutical preparationfor treating or preventing a condition characterized by retinaldegeneration, comprising an effective amount of RPE cells, which RPEcells are derived from human embryonic stem cells or other pluripotentstem cells. The pharmaceutical preparation may be formulated in apharmaceutically acceptable carrier according to the route ofadministration. For example, the preparation may be formulated foradministration to the subretinal space of the eye. The composition maycomprise at least 10⁴, 10⁵, 5×10⁵, 6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵, 10⁶,2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, or 10⁷ RPEcells. In certain embodiments, the composition may comprise at least2×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸ RPE cells. In certainembodiments, the RPE cells may include mature RPE cells, and thus thecell number includes the total of both differentiated RPE cells andmature differentiated RPE cells.

In another aspect, the invention provides a method for screening toidentify agents that modulate the survival of RPE cells. For example,RPE cells differentiated from human embryonic stem cells can be used toscreen for agents that promote RPE survival. Identified agents can beused, alone or in combination with RPE cells, as part of a treatmentregimen. Alternatively, identified agents can be used as part of aculture method to improve the survival of RPE cells differentiated invitro.

In another aspect, the invention provides a method for screening toidentify agents that modulate RPE cell maturity. For example, RPE cellsdifferentiated from human ES cells can be used to screen for agents thatpromote RPE maturation.

In certain embodiments of any of the foregoing, the method is performedin accordance with Good Manufacturing Practices (GMP). In certainembodiments of any of the foregoing, the human embryonic stem cells orother pluripotent stem cells from which the RPE cells are differentiatedwere derived in accordance with Good Manufacturing Practices (GMP). Incertain embodiments of any of the foregoing, the human embryonic stemcells from which the RPE cells are differentiated were derived from oneor more blastomere removed from an early stage embryo without destroyingthe remaining embryo.

In another aspect, the invention contemplates that, instead of humanembryonic stem cells, the starting material for producing RPE cells, orpreparations thereof, can be other types of human pluripotent stemcells. By way of example, the invention contemplates that inducedpluripotent stem (iPS) cells are used as a starting material fordifferentiating RPE cells using the methods described herein. Such iPScells can be obtained from a cell bank, or otherwise previously derived.Alternatively, iPS cells can be newly generated prior to commencingdifferentiation to RPE cells.

In one embodiment, RPE cells or preparations differentiated frompluripotent stem cells, including iPS cells, are used in a therapeuticmethod.

The present invention also provides functional human retinal pigmentedepithelial cells (hRPEs) that are terminally differentiated from humanembryonic stem cells (hESCs) or other human pluripotent stem cells. Innon-human, primate transplantation experiments, these hRPEs can beidentified apart from other cells by means of their unique physicalcharacteristics, such as by their unique mRNA and protein expression.Moreover, when implanted into a validated animal model of retinaldegeneration, hRPEs may treat retinal degeneration in the diseasedanimal. Accordingly, the hRPEs of the invention are useful for treatingpatients afflicted by various retinal degenerative disorders. Thepresent invention therefore provides a renewable source of hRPEs thatcan be produced and manufactured under GLP-like and GMP-compliantconditions for the treatment of visual degenerative diseases anddisorders.

In certain embodiments, the present invention provides a human retinalpigmented epithelial cell derived from a human embryonic stem cell,which cell is pigmented and expresses at least one gene that is notexpressed in a cell that is not a human retinal pigmented epithelialcell. In certain embodiments, the human retinal pigmented epithelialcell is isolated from at least one protein, molecule, or other impuritythat is found in its natural environment.

In another embodiment, the invention provides a cell culture comprisinghuman RPE cells derived from human embryonic stem cells or otherpluripotent stem cells, which are pigmented and express at least onegene that is not expressed in a cell that is not a human RPE. When usedin this manner, pigmented refers to any level of pigmentation, forexample, the pigmentation that initial occurs when RPE cellsdifferentiate from ES cells. Pigmentation may vary with cell density andthe maturity of the differentiated RPE cells. However, when cells arereferred to as pigmented—the term is understood to refer to any and alllevels of pigmentation.

In some embodiments, the cell culture comprises a substantially purifiedpopulation of human RPE cells. A substantially purified population ofhRPE cells is one in which the hRPE cells comprise at least about 75% ofthe cells in the population. In other embodiments, a substantiallypurified population of hRPE cells is one in which the hRPE cellscomprise at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 97.5%, 98%, 99%, or even greater than 99% of the cells in thepopulation. In some embodiments, the pigmentation of the hRPE cells inthe cell culture is homogeneous. In other embodiments, the pigmentationof the hRPE cells in the cell culture is heterogeneous, and the cultureof RPE cells comprises both differentiated RPE cells and mature RPEcells. A cell culture of the invention may comprise at least about 10¹,10², 5×10², 10³, 5×10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, or at least about 10⁹hRPE cells.

The present invention provides human retinal pigmented epithelial cellswith varying degrees of pigmentation. In certain embodiments, thepigmentation of a human retinal pigmented epithelial cell is the same asan average human pigmented epithelial cell after terminaldifferentiation of the hRPE cell. In certain embodiments, thepigmentation of a human retinal pigmented epithelial cell is morepigmented than the average human retinal pigmented epithelial cell afterterminal differentiation of the hRPE cell. In certain other embodiments,the pigmentation of a human retinal pigmented epithelial cell is lesspigmented than the average human retinal pigmented epithelial cell afterterminal differentiation.

In certain embodiments, the present invention provides human RPE cellsdifferentiated from ES cells or other pluripotent stem cells and thatexpress (at the mRNA and/or protein level) one or more (1, 2, 3, 4, 5,or 6) of the following: RPE-65, Bestrophin, PEDF, CRALBP, Otx2, andMit-F. In certain embodiments, gene expression is measured by mRNAexpression. In other embodiments, gene expression is measured by proteinexpression. In certain embodiments, the RPE cells do not substantiallyexpress ES-specific genes, such as Oct-4, alkaline phosphatase, nanog,and/or Rex-1. In other embodiments, the RPE cells express one or more(1, 2, or 3) of pax-2, pax-6, and/or tyrosinase. In certain embodiments,expression of pax-2, pax-6, and/or tyrosinase distinguishesdifferentiated RPE cells from mature differentiated RPE cells. In otherembodiments, the RPE cells express one or more of the markers presentedin Table 2, and the expression of the one or more markers is upregulatedin RPE cells relative to expression (if any) in human ES cells. In otherembodiments, the RPE cells express one or more of the markers presentedin Table 3, and the expression of the one or more markers isdownregulated in RPE cells relative to expression (if any) in human EScells.

In certain embodiments, the invention provides a pharmaceuticalpreparation comprising human RPE cells derived from human embryonic stemcells or other pluripotent stem cells. Pharmaceutical preparations maycomprise at least about 10¹, 10², 5×10², 10³, 5×10³, 10⁴, 10⁵, 10⁶, 10⁷,10⁸ or about 10⁹ hRPE cells.

In other embodiments, the invention provides a cryopreserved preparationof the RPE cells described herein. The cryopreserved preparation may befrozen for storage for days or years. The cells may be stored by anyappropriate method known in the art, e.g., cryogenically frozen and maybe frozen at any temperature appropriate for storage of the cells. Forexample, the cells may be frozen at approximately −20° C., −80° C.,−120° C., or at any other temperature appropriate for storage of cells.Cryogenically frozen cells are stored in appropriate containers andprepared for storage to reduce risk of cell damage and maximize thelikelihood that the cells will survive thawing. In other embodiments,RPE cells can be maintained at room temperature, or refrigerated at, forexample, approximately 4° C. Cryopreserved preparations of thecompositions described herein may comprise at least about 10¹, 10²,5×10², 10³, 5×10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁷ or about 10⁹ hRPE cells. Incertain embodiments, the hRPE cells of the invention are recovered fromstorage following cryopreservation. In certain embodiments, greater than65%, 70%, 75,%, or greater than 80% of the RPE cells retain viabilityfollowing cryopreservation. In other embodiments, greater than 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or greater than 99% of the RPEcells retain viability following cryopreservation.

In another aspect, the invention provides substantially purifiedpreparations of human RPE cells have any combination of the structural,molecular, and functional characteristics described herein. Suchpreparations may be formulated as pharmaceutical preparations foradministration and/or may be formulated for cryopreservation.

In another aspect, the invention provides use of the human RPE cellsdescribed herein in the manufacture of a medicament to treat a conditionin a patient in need thereof. In another embodiment, the inventionprovides use of a cell culture comprising the human RPE cells describedherein in the manufacture of a medicament to treat a condition in apatient in need thereof. In another embodiment, the invention providesthe use of a pharmaceutical preparation comprising the human RPE cellsdescribed herein in the manufacture of a medicament to treat a conditionin a patient in need thereof. Conditions that may be treated include,without limitation, degenerative diseases of the retina, such asStargardt's macular dystrophy, retinitis pigmentosa, maculardegeneration, glaucoma, and diabetic retinopathy. In certainembodiments, the invention provides methods for treating or preventing acondition characterized by retinal degeneration, comprisingadministering to a subject in need thereof an effective amount of apreparation comprising RPE cells, which RPE cells are derived frommammalian embryonic stem cells. Conditions characterized by retinaldegeneration include, for example, Stargardt's macular dystrophy, agerelated macular degeneration, diabetic retinopathy, and retinitispigmentosa.

In other embodiments, the invention provides a solution of human RPEcells derived from a human embryonic stem cell, or other pluripotentstem cell, which RPE cells have any combinations of the featuresdescribed herein. Such a solutions may comprise at least about 10¹, 10²,5×10², 10³, 5×10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸ or about 10⁹ hRPE cells asdescribed herein. Such solutions are suitable for injection to asubject. Such solutions are suitable for cryopreservation as describedherein. This invention also provides a use of these solutions for themanufacture of a medicament to treat a disease that could be treated bythe administration of RPE cells, such as, for example, retinaldegenerative diseases of the eye.

In another aspect, the RPE cells of the invention are derived from humanembryonic stem cells, or other pluripotent stem cells, previouslyderived under GMP conditions. In one embodiment, the human ES cells arederived from one or more blastomeres of an early cleavage stage embryo,optionally without destroying the embryo. In another embodiment, thehuman ES cells are from a library of human embryonic stem cells. Incertain embodiments said library of human embryonic stem cells comprisesstem cells, each of which is hemizygous, homozygous, or nullizygous forat least one MHC allele present in a human population, wherein eachmember of said library of stem cells is hemizygous, homozygous, ornullizygous for a different set of MHC alleles relative to the remainingmembers of the library. In further embodiments, the library of humanembryonic stein cells comprises stem cells that are hemizygous,homozygous, or nullizygous for all MHC alleles present in a humanpopulation. In certain other embodiments, the invention provides alibrary of RPE cells, each of which is hemizygous, homozygous, ornullizygous for at least one MHC allele present in a human population,wherein each member of said library of RPE cells is hemizygous,homozygous, or nullizygous for a different set of MHC alleles relativeto the remaining members of the library. In further embodiments,invention provides a library of human RPE cells that are hemizygous,homozygous, or nullizygous for all MHC alleles present in a humanpopulation.

In certain embodiments of any of the foregoing, said substantiallypurified culture of RPE cells (with or without mature RPE cells) arefrozen for storage. The cells may be stored by any appropriate methodknown in the art, e.g., cryogenically frozen and may be frozen at anytemperature appropriate for storage of the cells. For example, the cellsmay be frozen at approximately −20° C., −80° C., −120° C., or at anyother temperature appropriate for storage of cells. Cryogenically frozencells are stored in appropriate containers and prepared for storage toreduce risk of cell damage and maximize the likelihood that the cellswill survive thawing. In other embodiments, RPE cells can be maintainedat room temperature, or refrigerated at, for example, approximately 4°C.

In certain embodiments of any of the foregoing, human RPE cells areproduced in accordance with Good Manufacturing Practices (GMP). Incertain embodiments of any of the foregoing, the human embryonic stemcells from which the RPE cells are differentiated were derived inaccordance with Good Manufacturing Practices (GMP). In certainembodiments of any of the foregoing, the human embryonic stem cells fromwhich the RPE cells are differentiated were derived from one or moreblastomeres removed from an early stage embryo without destroying theremaining embryo. As such, the invention provides GMP compliantpreparations of RPE cells, including substantially purified preparationsof RPE cells. Such preparations are substantially free of viral,bacterial, and/or fungal contamination or infection.

In certain embodiments of any of the foregoing, compositions orpreparations of RPE cells comprise at least 1×10³ RPE cells, at least5×10⁵ RPE cells, at least 1×10⁶ RPE cells, at least 5×10⁶ RPE cells, atleast 1×10⁷ RPE cells, at least 2×10⁷ RPE cells, at least 3×10⁷ RPEcells, at least 4×10⁷ RPE cells, at least 5×10⁷ RPE cells, at least6×10⁷ RPE cells, at least 7×10⁷ RPE cells, at least 8×10⁷ RPE cells, atleast 9×10⁷ RPE cells, at least 1×10⁸ RPE cells, at least 2×10⁸ RPEcells, at least 5×10⁸ RPE cells, at least 7×10⁸ RPE cells, or at least1×10⁹ RPE cells. In certain embodiments, the number of RPE cells in thepreparation includes differentiated RPE cells, regardless of level ofmaturity and regardless of the relative percentages of differentiatedRPE cells and mature differentiated RPE cells. In other embodiments, thenumber of RPE cells in the preparation refers to the number of eitherdifferentiated RPE cells or mature RPE cells.

In certain embodiments, the method further comprises formulating thedifferentiated RPE cells and/or differentiated mature RPE cells toproduce a preparation of RPE cells suitable for transplantation.

In another aspect, the invention provides a method for treating orpreventing a condition characterized by retinal degeneration, comprisingadministering to a subject in need thereof an effective amount of apreparation comprising RPE cells, which RPE cells are derived from humanpluripotent stem cells. In certain embodiments, the RPE cells arederived using any of the methods described herein. Conditionscharacterized by retinal degeneration include, for example, Stargardt'smacular dystrophy, age related macular degeneration (dry or wet),diabetic retinopathy, and retinitis pigmentosa.

In certain embodiments, the preparation was previously cryopreserved andwas thawed before transplantation.

In certain embodiments, the method of treating further comprisesadministration of cyclosporin or one or more other immunosuppressants.When immunosuppressants are used, they may be administered systemicallyor locally, and they may be administered prior to, concomitantly with,or following administration of the RPE cells. In certain embodiments,immunosuppressive therapy continues for weeks, months, years, orindefinitely following administration of RPE cells.

In certain embodiments, the method of treatment comprises administrationof a single dose of RPE cells. In other embodiments, the method oftreatment comprises a course of therapy where RPE cells are administeredmultiple times over some period. Exemplary courses of treatment maycomprise weekly, biweekly, monthly, quarterly, biannually, or yearlytreatments. Alternatively, treatment may proceed in phases wherebymultiple doses are required initially (e.g., daily doses for the firstweek), and subsequently fewer and less frequent doses are needed.Numerous treatment regimens are contemplated.

In certain embodiments, the administered RPE cells comprise a mixedpopulation of differentiated RPE cells and mature RPE cells. In otherembodiments, the administered RPE cells comprise a substantiallypurified population of either differentiated RPE cells or mature RPEcells. For example, the administered RPE cells may contain less than25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1% ofthe other RPE cell-type.

In certain embodiments, the RPE cells are formulated in apharmaceutically acceptable carrier or excipient.

In certain embodiments, the preparation comprising RPE cells istransplanted in a suspension, matrix or substrate. In certainembodiments, the preparation is administered by injection into thesubretinal space of the eye. In certain embodiments, the preparation isadministered transcomeally. In certain embodiments, about 10⁴ to about10⁶ cells are administered to the subject. In certain embodiments, themethod further comprises the step of monitoring the efficacy oftreatment or prevention by measuring electroretinogram responses,optomotor acuity threshold, or luminance threshold in the subject. Themethod may also comprise monitoring the efficacy of treatment orprevention by monitoring immunogenicity of the cells or migration of thecells in the eye.

In certain aspects, the invention provides a pharmaceutical preparationfor treating or preventing a condition characterized by retinaldegeneration, comprising an effective amount of RPE cells, which RPEcells are derived from human embryonic stem cells. The pharmaceuticalpreparation may be formulated in a pharmaceutically acceptable carrieraccording to the route of administration. For example, the preparationmay be formulated for administration to the subretinal space or corneaof the eye. The composition may comprise at least 10⁴, 10⁵, 5×10⁵,6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵, 10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶,7×10⁶, 8×10⁶, 9×10⁶, or 10⁷ RPE cells. In certain embodiments, thecomposition may comprise at least 2×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷,9×10⁷, 1×10⁸ RPE cells. In certain embodiments, the RPE cells mayinclude mature RPE cells, and thus the cell number includes the total ofboth differentiated RPE cells and mature differentiated RPE cells.

In another aspect, the invention provides a method for screening toidentify agents that modulate the survival of RPE cells. For example,RPE cells differentiated from human embryonic stem cells can be used toscreen for agents that promote RPE survival. Identified agents can beused, alone or in combination with RPE cells, as part of a treatmentregimen. Alternatively, identified agents can be used as part of aculture method to improve the survival of RPE cells differentiated invitro.

In another aspect, the invention provides a method for screening toidentify agents that modulate RPE cell maturity. For example, RPE cellsdifferentiated from human ES cells can be used to screen for agents thatpromote RPE maturation.

In certain embodiments of any of the foregoing, the method is performedin accordance with Good Manufacturing Practices (GMP). In certainembodiments of any of the foregoing, the human embryonic stem cells fromwhich the RPE cells are differentiated were derived in accordance withGood Manufacturing Practices (GMP). In certain embodiments of any of theforegoing, the human embryonic stem cells from which the RPE cells aredifferentiated were derived from one or more blastomere removed from anearly stage embryo without destroying the remaining embryo.

In another aspect, the invention contemplates that, instead of humanembryonic stem cells, the starting material for producing RPE cells, orpreparations thereof, can be other types of human pluripotent stemcells. By way of example, the invention contemplates that inducedpluripotent stem (iPS) cells, which have the characteristic of hES, cansimilarly be used as a starting material for differentiating RPE cellsusing the methods described herein. Such iPS cells can be obtained froma cell bank, or otherwise previously derived. Alternatively, iPS cellscan be newly generated prior to commencing differentiation to RPE cells.

In one embodiment, RPE cells or preparations differentiated frompluripotent stem cells, including iPS cells, are used in a therapeuticmethod.

The invention contemplates any combination of the aspects andembodiments described above or below. For example, preparations of RPEcells comprising any combination of differentiated RPE cells and matureRPE cells can be used in the treatment of any of the diseases andconditions described herein. Similarly, methods described herein forproducing RPE cells using human embryonic stem cells as a startingmaterial may be similarly performed using any human pluripotent stemcells as a starting material.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic model showing the developmental ontogeny of humanRPE cells derived from human embryonic stem cells.

FIG. 2 is a graph showing gene expression comparison of hES cells andhuman embryonic stem cell-derived RPE cells by quantitative, Real-Time,Reverse Transcription PCR (qPCR).

FIG. 3 is a graph showing gene expression comparison of ARPE-19 cells (apreviously derived RPE cell line) and human embryonic stem cell-derivedRPE cells by quantitative, Real-Time, Reverse Transcription PCR (qPCR).

FIG. 4 is a graph showing gene expression comparison of fetal RPE cellsand human embryonic stem cell-derived RPE cells by quantitative,Real-Time, Reverse Transcription PCR (qPCR).

FIG. 5 is a graph showing gene expression comparison of mature RPE cellsand hES cells by quantitative, Real-Time, Reverse Transcription PCR(qPCR).

FIG. 6 is a photomicrograph showing Western Blot analysis ofhES-specific and RPE-specific markers. Embryonic stem cell-derived RPEcells (lane 1) derived from hES cells (lane 2) did not express thehES-specific proteins Oct-4, Nanog, and Rex-1. However, embryonic stemcell-derived RPE cells express RPE-specific proteins included RPE65,CRALBP, PEDF, Bestrophin, PAX6, and Otx2. Actin was used as proteinloading control.

FIG. 7 is a graph showing the principal components analysis plot ofmicroarray gene expressions. Component 1, representing 69% of thevariability represents the cell type, whereas Component 2, representsthe cell line (i.e., genetic variability). A near-linear scatter of geneexpression profiles characterizes the developmental ontogeny of hRPEderived from hES cells.

DETAILED DESCRIPTION OF THE INVENTION

In order that the invention herein described may be fully understood,the following detailed description is set forth. Various embodiments ofthe invention are described in detail and may be further illustrated bythe provided examples.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as those commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe invention or testing of the present invention, suitable methods andmaterials are described below. The materials, methods and examples areillustrative only, and are not intended to be limiting.

All publications, patents, patent publications and applications andother documents mentioned herein are incorporated by reference in theirentirety.

In order to further define the invention, the following terms anddefinitions are provided herein.

As used in the description herein and throughout the claims that follow,the meaning of “a,” “an,” and “the” includes plural reference unless thecontext clearly dictates otherwise. Also, as used in the descriptionherein, the meaning of “in” includes “in” and “on” unless the contextclearly dictates otherwise.

Throughout this specification, the word “comprise” or variations such as“comprises” or “comprising” will be understood to imply the inclusion ofa stated integer or groups of integers but not the exclusion of anyother integer or group of integers.

By “embryo” or “embryonic” is meant a developing cell mass that has notimplanted into the uterine membrane of a maternal host. An “embryoniccell” is a cell isolated from or contained in an embryo. This alsoincludes blastomeres, obtained as early as the two-cell stage, andaggregated blastomeres.

The term “embryonic stem cells” refers to embryo-derived cells. Morespecifically it refers to cells isolated from the inner cell mass ofblastocysts or morulae and that have been serially passaged as celllines. The term also includes cells isolated from one or moreblastomeres of an embryo, preferably without destroying the remainder ofthe embryo. The term also includes cells produced by somatic cellnuclear transfer, even when non-embryonic cells are used in the process.

The term “human embryonic stem cells” (hES cells) is used herein as itis used in the art. This term includes cells derived from the inner cellmass of human blastocysts or morulae that have been serially passaged ascell lines. The hES cells may be derived from fertilization of an eggcell with sperm or DNA, nuclear transfer, parthenogenesis, or by meansto generate hES cells with homozygosity in the HLA region. Human EScells are also cells derived from a zygote, blastomeres, orblastocyst-staged mammalian embryo produced by the fusion of a sperm andegg cell, nuclear transfer, parthenogenesis, or the reprogramming ofchromatin and subsequent incorporation of the reprogrammed chromatininto a plasma membrane to produce a cell. Human embryonic stem cells ofthe present invention may include, but are not limited to, MA01, MA09,ACT-4, No. 3, H1, H7, H9, H14 and ACT30 embryonic stem cells. In certainembodiments, human ES cells used to produce RPE cells are derived andmaintained in accordance with GMP standards. Human embryonic stem cells,regardless of their source or the particular method use to produce them,can be identified based on (i) the ability to differentiate into cellsof all three germ layers, (ii) expression of at least Oct-4 and alkalinephosphatase, and (iii) ability to produce teratomas when transplantedinto immunocompromised animals.

The term “human embryo-derived cells” (hEDC) refers to morula-derivedcells, blastocyst-derived cells including those of the inner cell mass,embryonic shield, or epiblast, or other totipotent or pluripotent stemcells of the early embryo, including primitive endoderm, ectoderm, andmesoderm and their derivatives, also including blastomeres and cellmasses from aggregated single blastomeres or embryos from varying stagesof development, but excluding human embryonic stem cells that have beenpassaged as cell lines.

As used herein, the term “pluripotent stem cells” includes embryonicstem cells, embryo-derived stem cells, and induced pluripotent stemcells, regardless of the method by which the pluripotent stem cells arederived. Pluripotent stem cells are defined functionally as stem cellsthat: (a) are capable of inducing teratomas when transplanted inimmunodeficient (SCID) mice; (b) are capable of differentiating to celltypes of all three germ layers (e.g., can differentiate to ectodermal,mesodermal, and endodermal cell types); and (c) express one or moremarkers of embryonic stem cells (e.g., express Oct 4, alkalinephosphatase, SSEA-3 surface antigen, SSEA-4 surface antigen, nanog,TRA-1-60, TRA-1-81, SOX2, REX1, etc). Exemplary pluripotent stem cellscan be generated using, for example, methods known in the art. Exemplarypluripotent stem cells include embryonic stem cells derived from the ICMof blastocyst stage embryos, as well as embryonic stem cells derivedfrom one or more blastomeres of a cleavage stage or morula stage embryo(optionally without destroying the remainder of the embryo). Suchembryonic stem cells can be generated from embryonic material producedby fertilization or by asexual means, including somatic cell nucleartransfer (SCNT), parthenogenesis, and androgenesis. Further exemplarypluripotent stem cells include induced pluripotent stem cells (iPScells) generated by reprogramming a somatic cell by expressing orinducing expression of a combination of factors (herein referred to asreprogramming factors). iPS cells can be generated using fetal,postnatal, newborn, juvenile, or adult somatic cells. In certainembodiments, factors that can be used to reprogram somatic cells topluripotent stem cells include, for example, a combination of Oct4(sometimes referred to as Oct 3/4), Sox2, c-Myc, and KIf4. In otherembodiments, factors that can be used to reprogram somatic cells topluripotent stem cells include, for example, a combination of Oct 4,Sox2, Nanog, and Lin28. In other embodiments, somatic cells arereprogrammed by expressing at least 2 reprogramming factors, at leastthree reprogramming factors, or four reprogramming factors. In otherembodiments, additional reprogramming factors are identified and usedalone or in combination with one or more known reprogramming factors toreprogram a somatic cell to a pluripotent stem cell.

The terms “RPE cell” and “differentiated RPE cell” and “ES-derived RPEcell” and “human RPE cell” are used interchangeably throughout to referto an RPE cell differentiated from a human embryonic stem cell using amethod of the invention. The term is used generically to refer todifferentiated RPE cells, regardless of the level of maturity of thecells, and thus may encompass RPE cells of various levels of maturity.Differentiated RPE cells can be visually recognized by their cobblestonemorphology and the initial appearance of pigment. Differentiated RPEcells can also be identified molecularly based on substantial lack ofexpression of embryonic stem cell markers such as Oct-4 and nanog, aswell as based on the expression of RPE markers such as RPE-65, PEDF,CRALBP, and bestrophin. Note that when other RPE-like cells are referredto, they are generally referred to specifically as adult, fetal orAPRE19 cells. Thus, unless otherwise specified, RPE cells, as usedherein, refers to RPE cells differentiated from human embryonic stemcells.

The terms “mature RPE cell” and “mature differentiated RPE cell” areused interchangeably throughout to refer to changes that occur followinginitial differentiating of RPE cells. Specifically, although RPE cellscan be recognized, in part, based on initial appearance of pigment,after differentiation mature RPE cells can be recognized based onenhanced pigmentation. Pigmentation post-differentiation is notindicative of a change in the RPE state of the cells (e.g., the cellsare still differentiated RPE cells). Rather, the changes in pigmentpost-differentiation correspond to the density at which the RPE cellsare cultured and maintained. Thus, mature RPE cells have increasedpigmentation that accumulates after initial differentiation. Mature RPEcells are more pigmented than RPE cells—although RPE cells do have somelevel of pigmentation. Mature RPE cells can be subcultured at a lowerdensity, such that the pigmentation decreases. In this context, matureRPE cells can be cultured to produce RPE cells. Such RPE cells are stilldifferentiated RPE cells that express markers of RPE differentiation.Thus, in contrast to the initial appearance of pigmentation that occurswhen RPE cells begin to differentiate, pigmentation changespost-differentiation are phenomenological and do not reflectdedifferentiation of the cells away from an RPE fate. Note that changesin pigmentation post-differentiation also correlate with changes inpax-2 expression. Note that when other RPE-like cells are referred to,they are generally referred to specifically as adult, fetal or APRE19cells. Thus, unless otherwise specified, RPE cells, as used herein,refers to RPE cells differentiated from human embryonic stem cells.

“APRE-19” refers to cells of a previously derived, human RPE cell line.APRE-19 cells arose spontaneously and are not derived from human embryosor embryonic stein cells.

Overview

This invention provides preparations and compositions comprising humanretinal pigmented epithelium (RPE) cells derived from human embryonicstem cells or other human pluripotent stem cells. The RPE cells arepigmented, to at least some extent. The RPE cells do not express (at anyappreciable level) the embryonic stem cell markers Oct-4, nanog, orRex-1. Specifically, expression of these ES genes is approximately100-1000 fold lower in RPE cells than in ES cells, when assessed byquantitative RT-PCR. The RPE cells do express, both at the mRNA andprotein level, one or more of the following: RPE65, CRALBP, PEDF,Bestrophin, MitF and/or Otx2. In certain other embodiments, the RPEcells express, both at the mRNA and protein level, one or more of Pax-2,Pax-6, MitF, and tyrosinase. In certain embodiments of any of theforegoing, the RPE cells are mature RPE cells with increasedpigmentation in comparison to differentiated RPE cells.

The invention provides for human RPE cells, cell cultures comprising asubstantially purified population of human RPE cells, pharmaceuticalpreparations comprising human retinal pigmented epithelial cells andcryopreserved preparations of the human RPE cells. In certainembodiments, the invention provides for the use of the human RPE cellsin the manufacture of a medicament to treat a condition in a patient inneed thereof. Alternatively, the invention provides the use of the cellcultures in the manufacture of a medicament to treat a condition in apatient in need thereof. The invention also provides the use of thepharmaceutical preparations in the manufacture of a medicament to treata condition in a patient in need thereof. In any of the foregoing,preparations comprising RPE cells may include differentiated RPE cellsof varying levels of maturity, or may be substantially pure with respectto differentiated RPE cells of a particular level of maturity. Incertain embodiments of any of the foregoing, the preparations comprisingRPE cells are prepared in accordance with Good Manufacturing Practices(GMP) (e.g., the preparations are GMP-compliant). In certainembodiments, the preparations comprising RPE cells are substantiallyfree of bacterial, viral, or fungal contamination or infection.

The human RPE cells (embryo-derived or derived from other pluripotentstem cells) can be identified and characterized based on theirstructural properties. Specifically, and in certain embodiments, thesecells are unique in that they can be identified or characterized basedon the expression or lack of expression (as assessed at the level of thegene or the level of the protein) of one or more markers. For example,in certain embodiments, differentiated ES-derived RPE cells can beidentified or characterized based on expression of one or more (e.g.,the cells can be characterized based on expression of at least one, atleast two, at least three, at least four, at least five, or at leastsix) of the following markers: RPE-65, Bestrophin, PEDF, CRALBP, Otx2,and Mit-F. Additionally or alternatively, ES-derived RPE cells can beidentified or characterized based on expression of PAX2, tyrosinase,and/or PAX6. Additionally or alternatively, hRPE cells can be identifiedor characterized based on expression or lack of expression (as assessedat the level of the gene or the level of the protein) of one or more (1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) markers analyzed in any ofTables 1-3.

Additionally or alternatively, ES-derived RPE cells can also beidentified and characterized based on the degree of pigmentation of thecell. Differentiated hRPE cells that are rapidly dividing are lightlypigmented. However, when cell density reaches maximal capacity, or whenhRPE cells are specifically matured, hRPE take on their characteristicphenotypic hexagonal shape and increase pigmentation level byaccumulating melanin and lipofuscin. As such, initial accumulation ofpigmentation serves as an indicator of RPE differentiation and increasedpigmentation associated with cell density serves as an indicator of RPEmaturity.

Preparations comprising RPE cells include preparations that aresubstantially pure, with respect to non-RPE cell types, but whichcontain a mixture of differentiated RPE cells and mature differentiatedRPE cells. Preparations comprising RPE cells also include preparationsthat are substantially pure both respect to non-RPE cell types and withrespect to RPE cells of other levels of maturity.

For any of the foregoing embodiments, the invention contemplates thatthe RPE cells (characterized as described above) may be derived fromhuman pluripotent stem cells, for example iPS cells and embryonic stemcells. In certain embodiments, the RPE cells are derived from humanpluripotent stem cells using any of the methods described herein.

RPE Cell Differentiation

Embryonic stem cells (ES) can be indefinitely maintained in vitro in anundifferentiated state and yet are capable of differentiating intovirtually any cell type, providing a limitless supply of rejuvenated andhistocompatible cells for transplantation therapy. The problem of immunerejection can be overcome with nuclear transfer and parthenogenetictechnology. Thus, human embryonic stem (hES) cells are useful forstudies on the differentiation of human cells and can be considered as apotential source for transplantation therapies. To date, thedifferentiation of human and mouse ES cells into numerous cell typeshave been reported (reviewed by Smith, 2001) including cardiomyocytes[Kehat et al. 2001, Mummery et al., 2003 Carpenter et al., 2002],neurons and neural precursors (Reubinoff et al. 2000, Carpenter et al.2001, Schuldiner et al., 2001), adipocytes (Bost et al., 2002, Aubert etal., 1999), hepatocyte-like cells (Rambhatla et al., 2003), hematopoeticcells (Chadwick et al., 2003). oocytes (Hubner et al., 2003),thymocyte-like cells (Lin R Y et al., 2003), pancreatic islet cells(Kahan, 2003), and osteoblasts (Zur Nieden et al., 2003).

The present invention provides for the differentiation of human ES cellsinto a specialized cell in the neuronal lineage, the retinal pigmentepithelium (RPE). RPE is a densely pigmented epithelial monolayerbetween the choroid and neural retina. It serves as a part of a barrierbetween the bloodstream and retina. Its functions include phagocytosisof shed rod and cone outer segments, absorption of stray light, vitaminA metabolism, regeneration of retinoids, and tissue repair (Grierson etal., 1994, Fisher and Reh, 2001, Marmorstein et al., 1998). The RPE canbe recognized by its cobblestone cellular morphology of black pigmentedcells. In addition, there are several known markers of the RPE,including cellular retinaldchyde-binding protein (CRALBP), a cytoplasmicprotein that is also found in apical microvilli (Bunt-Milam and Saari,1983); RPE65, a cytoplasmic protein involved in retinoid metabolism (Maet al., 2001, Redmond et al., 1998); bestrophin, the product of the Bestvitelliform macular dystrophy gene (VMD2, Marmorstein et al., 2000), andpigment epithelium derived factor (PEDF), a 48 kD secreted protein withangiostatic properties (Karakousis et al., 2001, Jablonski et al.,2000).

RPE plays an important role in photoreceptor maintenance, and variousRPE malfunctions in vivo are associated with a number of vision-alteringailments, such as RPE detachment, displasia, atrophy, retinopathy,retinitis pigmentosa, macular dystrophy or degeneration, includingage-related macular degeneration, which can result in photoreceptordamage and blindness. Because of its wound healing abilities, RPE hasbeen extensively studied in application to transplantation therapy. Ithas been shown in several animal models and in humans (Gouras et al.,2002, Stanga et al., 2002, Binder et al., 2002, Schraermeyer et al.,2001, reviewed by Lund et al., 2001) that RPE transplantation has a goodpotential of vision restoration. Recently another prospective niche forRPE transplantation was proposed and even reached the phase of clinicaltrials: since these cells secrete dopamine, they could be used fortreatment of Parkinson disease (Subramanian, 2001). However, even in animmune-privileged eye, there is a problem of graft rejection, hinderingthe progress of this approach if allogenic transplant is used. The otherproblem is the reliance on fetal tissue, as adult RPE has a very lowproliferative potential. The present invention decreases the likelihoodthat graft rejection will occur and removes the reliance on the use offetal tissue.

As a source of immune compatible tissues, hES cells hold a promise fortransplantation therapy, as the problem of immune rejection can beovercome with nuclear transfer technology. The use of the newdifferentiation derivatives of human ES cells, including retinal pigmentepithelium-like cells and neuronal precursor cells, and the use of thedifferentiation system for producing the same offers an attractivepotential supply of RPE and neuronal precursor cells fortransplantation.

Accordingly, one aspect of the present invention is to provide animproved method of generating RPE cells derived from human embryonicstem cells, which may be purified and/or isolated. Such cells are usefulfor therapy for retinal degeneration diseases such as, for example,retinitis pigmentosa, macular degeneration and other eye disorders. Thecell types that can be produced using this invention include, but arenot limited to, RPE cells and RPE progenitor cells. Cells that may alsobe produced include iris pigmented epithelial (IPE) cells and othervision associated neural cells, such as internuncial neurons (e.g.“relay” neurons of the inner nuclear layer (INL)) and amacrine cells.Additionally, retinal cells, rods, cones, and corneal cells can beproduced. In another embodiment of the present invention, cellsproviding the vasculature of the eye can also be produced.

The human embryonic stem cells are the starting material of this method.The embryonic stem cells may be cultured in any way known in the art,such as in the presence or absence of feeder cells. Additionally, humanES cells produced using any method can be used as the starting materialto produce RPE cells. For example, the human ES cells may be derivedfrom blastocyst stage embryos that were the product of in vitrofertilization of egg and sperm. Alternatively, the human ES cells may bederived from one or more blastomeres removed from an early cleavagestage embryo, optionally, without destroying the remainder of theembryo. In still other embodiments, the human ES cells may be producedusing nuclear transfer. As a starting material, previously cryopreservedhuman ES cells can be used.

In the first step of this method for producing RPE cells, humanembryonic stem cells are cultured as embryoid bodies. Embryonic stemcells may be pelleted, resuspended in culture medium, and plated onculture dishes (e.g., low attachment culture dishes). Cells may becultured in any medium that is sufficient for growth of cells athigh-density, such as, commercially available medium for viral,bacterial, insect, or animal cell culture. In certain embodiments,nutrient rich, low protein medium is used (e.g., MDBK-GM medium,containing about 150 mg/mL (0.015%) animal-derived protein). When lowprotein medium is used, the medium contains, for example, less than orequal to about 5%, 4%, 3%, 2.5%, 2%, 1.5%, 1%, 0.75%, 0.5%, 0.25%, 0.2%,0.1%, 0.05%, 0.02%, 0.016%, 0.015%, or even less than or equal to 0.010%animal-derived protein. Note that reference to the percentage of proteinpresent in low protein medium refers to the medium alone and does notaccount for protein present in, for example, B-27 supplement. Thus, itis understood that when cells are cultured in low protein medium andB-27 supplement, the percentage of protein present in the medium may behigher.

In certain embodiments, nutrient rich, protein-free medium is used(e.g., MDBK-MM medium). Other examples of culture media include, forexample, OptiPro SFM, VP-SFM, and EGM-2. Such media may include nutrientcomponents such as insulin, transferrin, sodium selenite, glutamine,albumin, ethanolamine, fetuin, peptone, purified lipoprotein material,vitamin A, vitamin C, and vitamin E.

In certain embodiments, cell cultures in either low protein or proteinfree medium are supplemented with serum free B-27 supplement (Brewer etal., Journal of Neuroscience Research 35:567-576 (1993)). Nutrientcomponents of B27 supplement may include biotin, L-carnitine,corticosterone, ethanolamine, D+-galactose, reduced glutathione,lineleic acid, linolenic acid, progesterone, putrescine, retinylacetate, selenium, triodo-1-thyronine (T3), DL-alpha-tocopherol (vitaminE), DL-alpha-tocopherol acedate, bovine serum albumin, catalase,insulin, superoxide dismutase, and transferrin. When cells are culturedin protein free medium supplemented with B-27, protein free refers tothe medium prior to addition of B-27.

The medium may also contain supplements such as heparin, hydrocortisone,ascorbic acid, serum (such as, for example, fetal bovine scrum), or agrowth matrix (such as, for example, extracellular matrix from bovinecorneal epithelium, matrigel (BD biosciences), or gelatin).

In this method of the present invention, RPE cells differentiate fromthe embryoid bodies. Isolating RPE cells from the EBs allows for theexpansion of the RPE cells in an enriched culture in vitro. For humancells, RPE cells may be obtained form EBs grown for less than 90 days.In certain embodiments of the present invention, RPE cells arise inhuman EBs grown for 7-14 days. In other embodiments, RPE cells arise inhuman EBs grown for 14-28 days. In another embodiment, RPE cells areidentified and may be isolated from human EBs grown for 28-45 days. Inanother embodiment, RPE cells arise in human EBs grown for 45-90 days.The medium used to culture embryonic stem cells, embryoid bodies, andRPE cells may be removed and/or replaced with the same or differentmedia at any interval. For example, the medium may be removed and/orreplaced after 0-7 days, 7-10 days, 10-14 days, 14-28 days, or 28-90days. In certain embodiments, the medium is replaced at least daily,every other day, or at least every three days.

In certain embodiments, the RPE cells that differentiate from the EBsare washed and dissociated (e.g., by Trypsin/EDTA, collegenase B,collegenase IV, or dispase). In certain embodiments, a non-enzymaticsolution is used to disassociate the cells, such as a highEDTA-containing solution such as, for example, Hanks-based celldisassociation buffer.

RPE cells are selected from the dissociated cells and culturedseparately to produce a substantially purified culture of RPE cells. RPEcells are selected based on characteristics associated with RPE cells.For example, RPE cells can be recognized by cobblestone cellularmorphology and pigmentation. In addition, there are several knownmarkers of the RPE, including cellular retinaldehyde-binding protein(CRALBP), a cytoplasmic protein that is also found in apical microvilli(Bunt-Milam and Saari, 1983); RPE65, a cytoplasmic protein involved inretinoid metabolism (Ma et al., 2001, Redmond et al., 1998); bestrophin,the product of the Best vitelliform macular dystrophy gene (VMD2,Marmorstein et al., 2000), and pigment epithelium derived factor (PEDF),a 48 kD secreted protein with angiostatic properties (Karakousis et al.,2001, Jablonski et al., 2000). Alternatively, RPE cells can be selectedbased on cell function, such as by phagocytosis of shed rod and coneouter segments, absorption of stray light, vitamin A metabolism,regeneration of retinoids, and tissue repair (Grierson et al., 1994,Fisher and Reh, 2001, Marmorstein et al., 1998). Evaluation may also beperformed using behavioral tests, fluorescent angiography, histology,tight junctions conductivity, or evaluation using electron microscopy.Another embodiment of the present invention is a method of identifyingRPE cells by comparing the messenger RNA transcripts of such cells withcells derived in-vivo. In certain embodiments, an aliquot of cells istaken at various intervals during the differentiation of embryonic stemcells to RPE cells and assayed for the expression of any of the markersdescribed above. These characteristic distinguish differentiated RPEcells.

RPE cell culture media may be supplemented with one or more growthfactors or agents. Growth factors that may be used include, for example,EGF, FGF, VEGF, and recombinant insulin-like growth factor. Other growthfactors that may be used in the present invention include 6Ckine(recombinant), activin A, AlphaA-interferon, alpha-interferon,amphiregulin, angiogenin, B-endothelial cell growth factor, betacellulin, B-interferon, brain derived neurotrophic factor, C10(recombinant), cardiotrophin-1, ciliary neurotrophic factor,cytokine-induced neutrophil chemoattractant-1, endothelial cell growthsupplement, eotaxin, epidermal growth factor, epithelial neutrophilactivating peptide-78, erythropoiten, estrogen receptor-alpha, estrogenreceptor-B, fibroblast growth factor (acidic/basic, heparin stabilized,recombinant), FLT-3/FLK-2 ligand (FLT-3 ligand), gamma-interferon, glialcell line-derived neurotrophic factor, Gly-His-Lys, granulocytecolony-stimulating factor, granulocyte macrophage colony-stimulatingfactor, GRO-alpha/MGSA, GRO-B, GRO-gamma, HCC-1, heparin-bindingepidermal growth factor like growth factor, hepatocyte growth factor,heregulin-alpha (EGF domain), insulin growth factor binding protein-1,insulin-like growth factor binding protein-1/IGF-1 complex, insulin-likegrowth factor, insulin-like growth factor II, 2.5S nerve growth factor(NGF), 7S-NGF, macrophage inflammatory protein-1B, macrophageinflammatory protein-2, macrophage inflammatory protein-3 alpha,macrophage inflammatory protein-3B, monocyte chemotactic protein-1,monocyte chemotactic protein-2, monocyte chemotactic protein-3,neurotrophin-3, neurotrophin-4, NGF-B (human or rat recombinant),oncostatin M (human or mouse recombinant), pituitary extract, placentagrowth factor, platelet-derived endothelial cell growth factor,platelet-derived growth factor, pleiotrophin, rantes, stem cell factor,stromal cell-derived factor 1B/pre-B cell growth stimulating factor,thrombopoetin, transforming growth factor alpha, transforming growthfactor-B1, transforming growth factor-B2, transforming growth factor-B3,transforming growth-factor-B5, tumor necrosis factor (alpha and B), andvascular endothelial growth factor. Agents that can be used according tothe present invention include cytokines such as interferon-alpha A,interferon-alpha A/D, interferon-.beta., interferon-gamma,interferon-gamma-inducible protein-10, interleukin-1, interleukin-2,interleukin-3, interleukin-4, interleukin-5, interleukin-6,interleukin-7, interleukin-8, interleukin-9, interleukin-10,interleukin-1, interleukin-12, interleukin-13, interleukin-15,interleukin-17, keratinocyte growth factor, leptin, leukemia inhibitoryfactor, macrophage colony-stimulating factor, and macrophageinflammatory protein-1 alpha.

Agents according to the invention also include hormones and hormoneantagonists, such as 17B-estradiol, adrenocorticotropic hormone,adrenomedullin, alpha-melanocyte stimulating hormone, chorionicgonadotropin, corticosteroid-binding globulin, corticosterone,dexamethasone, estriol, follicle stimulating hormone, gastrin 1,glucagon, gonadotropin, hydrocortisone, insulin, insulin-like growthfactor binding protein, L-3,3′,5′-triiodothyronine,L-3,3′,5-triiodothyronine, leptin, leutinizing hormone, L-thyroxine,melatonin, MZ-4, oxytocin, parathyroid hormone, PEC-60, pituitary growthhormone, progesterone, prolactin, secretin, sex hormone bindingglobulin, thyroid stimulating hormone, thyrotropin releasing factor,thyroxine-binding globulin, and vasopressin.

In addition, agents according to the invention include extracellularmatrix components such as fibronectin, proteolytic fragments offibronectin, laminin, thrombospondin, aggrecan, and syndezan.

Agents according to the invention also include antibodies to variousfactors, such as anti-low density lipoprotein receptor antibody,anti-progesterone receptor, internal antibody, anti-alpha interferonreceptor chain 2 antibody, anti-c-c chemokine receptor 1 antibody,anti-CD 118 antibody, anti-CD 119 antibody, anti-colony stimulatingfactor-1 antibody, anti-CSF-1 receptor/c-fins antibody, anti-epidermalgrowth factor (AB-3) antibody, anti-epidermal growth factor receptorantibody, anti-epidermal growth factor receptor, phospho-specificantibody, anti-epidermal growth factor (AB-1) antibody,anti-erythropoietin receptor antibody, anti-estrogen receptor antibody,anti-estrogen receptor, C-terminal antibody, anti-estrogen receptor-Bantibody, anti-fibroblast growth factor receptor antibody,anti-fibroblast growth factor, basic antibody, anti-gamma-interferonreceptor chain antibody, anti-gamma-interferon human recombinantantibody, anti-GFR alpha-1 C-terminal antibody, anti-GFR alpha-2C-terminal antibody, anti-granulocyte colony-stimulating factor (AB-1)antibody, anti-granulocyte colony-stimulating factor receptor antibody,anti-insulin receptor antibody, anti-insulin-like growth factor-1receptor antibody, anti-interleukin-6 human recombinant antibody,anti-interleukin-1 human recombinant antibody, anti-interleukin-2 humanrecombinant antibody, anti-leptin mouse recombinant antibody, anti-nervegrowth factor receptor antibody, anti-p60, chicken antibody,anti-parathyroid hormone-like protein antibody, anti-platelet-derivedgrowth factor receptor antibody, anti-platelet-derived growth factorreceptor-B antibody, anti-platelet-derived growth factor-alpha antibody,anti-progesterone receptor antibody, anti-retinoic acid receptor-alphaantibody, anti-thyroid hormone nuclear receptor antibody, anti-thyroidhormone nuclear receptor-alpha 1/Bi antibody, anti-transferrinreceptor/CD71 antibody, anti-transforming growth factor-alpha antibody,anti-transforming growth factor-B3 antibody, anti-tumor necrosisfactor-alpha antibody, and anti-vascular endothelial growth factorantibody.

Growth factors, agents, and other supplements described herein may beused alone or in combination with other factors, agents, or supplements.Factors, agents, and supplements may be added to the media immediatelyor any time after cell culture.

In certain embodiments, the RPE cells are further cultured to produce aculture of mature RPE cells. The medium used to culture the RPE cellscan be any medium appropriate for high-density cell culture growth, suchas described herein. For example, the cells described herein may becultured in VP-SFM, EGM-2, and MDBK-MM.

A more detailed description of certain operative combinations of theabove described features of the invention is provided below.

In certain embodiments, a previously derived culture of human embryonicstem cells is provided. The hES cells can be, for example, previouslyderived from a blastocyst (produced by fertilization or nucleartransfer) or from one or more blastomeres from an early cleavage stageembryo (optionally without destroying the remainder of the embryo). Thehuman ES cells are cultured as a suspension culture to produce embryoidbodies (EBs). The embryoid bodies are cultured in suspension forapproximately 7-14 days. However, in certain embodiments, the EBs can becultured in suspension for fewer than 7 days (less than 7, 6, 5, 4, 3,2, or less than 1 day) or greater than 14 days. The EBs can be culturedin medium optionally supplemented with B-27 supplement.

After culturing the EBs in suspension culture, the EBs can transferredto produce an adherent culture. For example, the EBs can be plated inmedium onto gelatin coated plates. When cultured as an adherent culture,the EBs can be cultured in the same type of media as when grown insuspension. In certain embodiments, the media is not supplemented withB-27 supplement when the cells are cultured as an adherent culture. Inother embodiments, the medium is supplemented with B-27 initially (e.g.,for less than or equal to about 7 days), but then subsequently culturedin the absence of B-27 for the remainder of the period as an adherentculture. The EBs can be cultured as an adherent culture forapproximately 14-28. However, in certain embodiments, the EBs can becultured for fewer than 14 days (less than 14, 13, 12, 11, 10, 9, 8, 7,6, 5, 4, 3, 2, or less than 1 day) or greater than 28 days.

RPE cells begin to differentiate from amongst cells in the adherentculture of EBs. RPE cells can be visually recognized based on theircobblestone morphology and the initial appearance of pigmentation. AsRPE cells continue to differentiate, clusters of RPE cells can beobserved.

To enrich for RPE cells and to establish substantially purified culturesof RPE cells, RPE cells are dissociated from each other and from non-RPEcells using mechanical and/or chemical methods. A suspension of RPEcells can then be transferred to fresh medium and a fresh culture vesselto provide an enriched population of RPE cells.

Enriched cultures of RPE cells can be cultured in appropriate medium,for example, EGM-2 medium. This, or a functionally equivalent or similarmedium, may be supplemented with one or more growth factors or agents(e.g., bFGF, heparin, hydrocortisone, vascular endothelial growthfactor, recombinant insulin-like growth factor, ascorbic acid, humanepidermal growth factor).

For embodiments in which the RPE cells are matured, the RPE cells can befurther cultured in, for example MDBK-MM medium until the desired levelof maturation is obtained. This can be determined by monitoring theincrease in pigmentation level during maturation. As an alternative toMDBK-MM medium, a functionally equivalent or similar medium, may beused. Regardless of the particular medium used to mature the RPE cells,the medium may optionally be supplemented with one or more growthfactors or agents.

The culture of RPE cells, and thus the preparations of RPE cellsprepared from these cultures, can be substantially pure RPE cellscontaining less than 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,or less than 1% non-RPE cells. In certain embodiments, the substantiallypurified (with respect to non-RPE cells) cultures contain RPE cells ofvarying levels of maturity. In other embodiments, the cultures aresubstantially pure both with respect to non-RPE cells and with respectto RPE cells of differing level of maturity.

For any of the foregoing embodiments, the invention contemplates thatthe RPE cells (characterized as described above) may be derived fromhuman pluripotent stem cells, for example iPS cells and embryonic stemcells. In certain embodiments, the RPE cells are derived from humanpluripotent stem cells using any of the methods described herein.

Preparations of Differentiated Pluripotent Stem Cell-Derived RPE Cells

The present invention provides preparations of human pluripotent stemcell-derived RPE cells. In certain embodiments, the preparation is apreparation of human embryonic stem cell-derived RPE cells. In certainembodiments, the preparation is a preparation of human iPS cell-derivedRPE cells. In certain embodiments, the preparations are substantiallypurified (with respect to non-RPE cells) preparations comprisingdifferentiated ES-derived RPE cells. By substantially purified, withrespect to non-RPE cells, is meant that the preparation comprises atleast 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or even greater than 99% RPE cells. In other words, the substantiallypurified preparation of RPE cells contains less than 25%, 20%, 15%, 10%,9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1% non-RPE cell type. Incertain embodiments, the RPE cells in such a substantially purifiedpreparation contain RPE cells of varying levels ofmaturity/pigmentation. In other embodiments, the RPE cells aresubstantially pure, both with respect to non-RPE cells and with respectto RPE cells of other levels of maturity. In certain embodiments, thepreparations are substantially purified, with respect to non-RPE cells,and enriched for mature RPE cells. By enriched for mature RPE cells, itis meant that at least 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even greaterthan 99% of the RPE cells are mature RPE cells. In other embodiments,the preparations are substantially purified, with respect to non-RPEcells, and enriched for differentiated RPE cells rather than mature RPEcells. By enriched for, it is meant that at least 30%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or even greater than 99% of the RPE cells aredifferentiated RPE cells rather than mature RPE cells. In certainembodiments, mature RPE cells are distinguished from RPE cells by one ormore of: the level of pigmentation, level of expression of Pax-2, Pax-6,and/or tyrosinase. In certain embodiments, the preparations include atleast 1×10³ RPE cells, 5×10³ RPE cells, 1×10⁴ RPE cells, 5×10⁴ RPEcells, 1×10⁵ RPE cells, 2×10⁵ RPE cells, 3×10⁵ RPE cells, 4×10⁵ RPEcells, 5×10⁵ RPE cells, 6×10⁵ RPE cells, 7×10⁵ RPE cells, 8×10⁵ RPEcells, 9×10⁵ RPE cells, 1×10⁶ RPE cells, 5×10⁶ RPE cells, 6×10⁶ RPEcells, 7×10⁶ RPE cells, 8×10⁶ RPE cells, 9×10⁶ RPE cells, 1×10⁷ RPEcells, 5×10⁷ RPE cells, 1×10⁸ RPE cells, 1×10⁹ RPE cells, or even morethan 1×10⁹ RPE cells.

In certain embodiments, the ES-derived RPE cells do not express ES cellmarkers. For example, expression of the ES cell genes Oct-4, nanog,and/or Rex-1 is approximately 100-1000 fold lower in RPE cells than inES cells, as assessed by quantitative RT-PCR. Thus, in comparison to EScells, RPE cells are substantially negative for Oct-4, nanog, and/orRex-1 gene expression.

In certain embodiments, the ES-derived RPE cells express, at the mRNAand protein level, one or more of the following: RPE65, bestrophin,PEDF, CRALBP, Otx2, and MitF. In certain embodiments, RPE cells expresstwo or more, three or more, four or more, five or more, or six of thesemarkers. In certain embodiments, the RPE cells additionally oralternatively express, at the mRNA and protein level, one or more (1, 2,or 3) of the following: pax-2, pax6, and tyrosinase. In otherembodiments, the level of maturity of the RPE cells is assessed byexpression of one or more (1, 2, or 3) of pax-2, pax6, and tyrosinase.

In certain embodiments, the ES-derived RPE cells express, at the mRNAand/or protein level, one or more (1, 2, 3, 4, 5, 6, 7, 8, or 9) of theRPE-specific genes listed in Table 1 (pax-6, pax-2, RPE65, PEDF, CRALBP,bestrophin, mitF, Otx-2, and tyrosinase, as well as one or more (1, 2,3, or 4) of the neuroretina genes listed in Table 1 (CHX10, NCAM,nestin, beta-tubulin). However, the RPE cells do not substantiallyexpress the ES cell specific genes Oct-4, nanog, and/or Rex-1 (e.g.,expression of the ES cell specific genes is 100-1000 fold less in RPEcells, as determined by quantitative RT-PCR).

In certain embodiments, the ES-derived RPE cells express, at the mRNAand/or protein level, one or more (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, or more than 48) of the genes listed in Table 2, and the expressionof the one or more genes is increased in RPE cells relative to the levelof expression (if any) in human ES cells. Alternatively or additionally,the ES-derived RPE cells express, at the mRNA and/or protein level oneor more (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, or more than 25) of the genes listed in Table 3,but the expression of the one or more genes is decreased (includingdecreased to nearly undetectable levels) in RPE cells relative to thelevel of expression in human ES cells.

In certain embodiments, the substantially purified preparation of RPEcells comprises RPE cells of differing levels of maturity (e.g.,differentiated RPE cells and mature differentiated RPE cells). In suchinstances, there may be variability across the preparation with respectto expression of markers indicative of pigmentation. For example,although such RPE cells may have substantially the same expression ofRPE65, PEDF, CRALBP, and bestrophin. The RPE cells may vary, dependingon level of maturity, with respect to expression of one or more ofpax-2, pax-6, mitF, and/or tyrosinase.

In certain embodiments, the ES-derived RPE cells are stable, terminallydifferentiated RPE cells that do not de-differentiate to a non-RPE celltype. In certain embodiments, the ES-derived RPE cells are functionalRPE cells.

In certain embodiments, the ES-derived RPE cells are characterized bythe ability to integrate into the retina upon corneal, sub-retinal, orother transplantation or administration into an animal.

The preparations are produced in compliance with GMP standards. As such,in certain embodiments, the preparations are GMP compliant preparations.In other embodiments, the preparations are substantially free of viral,bacterial, and/or fungal infection and contamination.

In certain embodiments, the preparations are cryopreserved for storageand future use. Thus, the invention provides cryopreserved preparationscomprising substantially purified RPE cells. Cryopreserved preparationsare formulated in excipients suitable to maintain cell viability duringand following cryopreservation. In certain embodiments, thecryopreserved preparation comprises at least 1×10³ RPE cells, 5×10³ RPEcells, 1×10⁴ RPE cells, 5×10⁴ RPE cells, 1×10⁵ RPE cells, 2×10⁵ RPEcells, 3×10⁵ RPE cells, 4×10⁵ RPE cells, 5×10⁵ RPE cells, 6×10⁵ RPEcells, 7×10⁵ RPE cells, 8×10⁵ RPE cells, 9×10⁵ RPE cells, 1×10⁶ RPEcells, 5×10⁶ RPE cells, 6×10⁶ RPE cells, 7×10⁶ RPE cells, 8×10⁶ RPEcells, 9×10⁶ RPE cells, 1×10⁷ RPE cells, 5×10⁷ RPE cells, 1×10⁸ RPEcells, 1×10⁹ RPE cells, or even more than 1×10⁹ RPE cells. Cryopreservedpreparations may have the same levels of purity with respect to non-RPEcells and/or with respect to RPE cells of varying levels of maturity asdetailed above. In certain embodiments, at least 65% of the RPE cells ina cryopreserved preparation of RPE cells retain viability followingthawing. In other embodiments, at least 70%, 75%, 80%, 85%, 90%, 81%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% of the RPEcells in a cryopreserved preparation of RPE cells retain viabilityfollowing thawing.

The RPE cells provided herein are human cells. Note, however, that thehuman cells may be used in human patients, as well as in animal modelsor animal patients. For example, the human cells may be tested in rat,dog, or non-human primate models of retinal degeneration. Additionally,the human cells may be used therapeutically to treat animals in needthereof, such as in a veterinary medical setting.

Preparations may be formulated as pharmaceutical preparations preparedin a pharmaceutically acceptable carrier or excipient. Preferredpreparations are specifically formulated for administration to the eye(e.g., sub-retinal, corneal, ocular, etc.)

In certain embodiments of any of the foregoing, the RPE cells arederived from human pluripotent stem cells, such as human embryonic stemcells or human iPS cells. The invention contemplates that any of thepreparations described herein may be derived from an appropriate humanpluripotent stem cell.

Preparations including one or more of any of the foregoing features arecontemplated.

The invention contemplates that any of the foregoing preparations of RPEcells, including substantially purified preparations and preparationshave a particular minimal number of RPE cells, may be used in thetreatment of any of the indications described herein. Further, RPE cellsdifferentiated using any of the methods described herein may be used inthe treatment of any of the indications described herein.

RPE Cell-Based Therapies

RPE cells and pharmaceutically preparations comprising RPE cellsproduced by the methods described herein and/or having thecharacteristics of RPE cell preparations described herein may be usedfor cell-based treatments in which RPE cells are needed or would improvetreatment. The following section describes methods of using RPE cellsprovided by the present invention for treating various conditions thatmay benefit from RPE cell-based therapies. The particular treatmentregimen, route of administration, and any adjuvant therapy will betailored based on the particular condition, the severity of thecondition, and the patient's overall health. Additionally, in certainembodiments, administration of RPE cells may be effective to fullyrestore any vision loss or other symptoms. In other embodiments,administration of RPE cells may be effective to reduce the severity ofthe symptoms and/or to prevent further degeneration in the patient'scondition. The invention contemplates that administration of apreparation comprising RPE cells can be used to treat (includingreducing the severity of the symptoms, in whole or in part) any of theforegoing or following conditions. Additionally, RPE cell administrationmay be used to help treat the symptoms of any injury to the endogenousRPE layer.

The invention contemplates that RPE cells, including preparationscomprising RPE cells, derived using any of the methods described hereincan be used in the treatment of any of the indications described herein.Further, the invention contemplates that any of the preparationscomprising RPE cells described herein can be used in the treatment ofany of the indications described herein.

Retinitis pigmentosa is a hereditary condition in which the visionreceptors are gradually destroyed through abnormal genetic programming.Some forms cause total blindness at relatively young ages, where otherforms demonstrate characteristic “bone spicule” retinal changes withlittle vision destruction. This disease affects some 1.5 million peopleworldwide. Two gene defects that cause autosomal recessive retinitispigmentosa have been found in genes expressed exclusively in RPE. One isdue to an RPE protein involved in vitamin A metabolism (cisretinaldehyde binding protein). The second involves another proteinunique to RPE, RPE65. This invention provides methods and compositionsfor treating both of these forms of retinitis pigmentosa byadministration of RPE cells.

In another embodiment, the present invention provides methods andcompositions for treating disorders associated with retinaldegeneration, including macular degeneration.

A further aspect of the present invention is the use of RPE cells forthe therapy of eye diseases, including hereditary and acquired eyediseases. Examples of acquired or hereditary eye diseases areage-related macular degeneration, glaucoma and diabetic retinopathy.

Age-related macular degeneration (AMD) is the most common reason forlegal blindness in western countries. Atrophy of the submacular retinalpigment epithelium and the development of choroidal neovascularizations(CNV) results secondarily in loss of central visual acuity. For themajority of patients with subfoveal CNV and geographic atrophy there. isat present no treatment available to prevent loss of central visualacuity. Early signs of AMD are deposits (druses) between retinal pigmentepithelium and Bruch's membrane. During the disease there is sproutingof choroid vessels into the subretinal space of the macula. This leadsto loss of central vision and reading ability.

Glaucoma is the name given to a group of diseases in which the pressurein the eye increases abnormally. This leads to restrictions of thevisual field and to the general diminution in the ability to see. Themost common form is primary glaucoma; two forms of this aredistinguished: chronic obtuse-angle glaucoma and acute angle closure.Secondary glaucoma may be caused by infections, tumors or injuries. Athird type, hereditary glaucoma, is usually derived from developmentaldisturbances during pregnancy. The aqueous humor in the eyeball is undera certain pressure which is necessary for the optical properties of theeye. This intraocular pressure is normally 15 to 20 millimeters ofmercury and is controlled by the equilibrium between aqueous productionand aqueous outflow. In glaucoma, the outflow of the aqueous humor inthe angle of the anterior chamber is blocked so that the pressure insidethe eye rises. Glaucoma usually develops in middle or advanced age, buthereditary forms and diseases are not uncommon in children andadolescents. Although the intraocular pressure is only slightly raisedand there are moreover no evident symptoms, gradual damage occurs,especially restriction of the visual field. Acute angle closure bycontrast causes pain, redness, dilation of the pupils and severedisturbances of vision. The cornea becomes cloudy, and the intraocularpressure is greatly increased. As the disease progresses, the visualfield becomes increasingly narrower, which can easily be detected usinga perimeter, an ophthalmologic instrument. Chronic glaucoma generallyresponds well to locally administered medicaments which enhance aqueousoutflow. Systemic active substances are sometimes given to reduceaqueous production. However, medicinal treatment is not alwayssuccessful. If medicinal therapy fails, laser therapy or conventionaloperations are used in order to create a new outflow for the aqueoushumor. Acute glaucoma is a medical emergency. If the intraocularpressure is not reduced within 24 hours, permanent damage occurs.

Diabetic retinopathy arises in cases of diabetes mellitus. It can leadto thickening of the basal membrane of the vascular endothelial cells asa result of glycosilation of proteins. It is the cause of early vascularsclerosis and the formation of capillary aneurysms. These vascularchanges lead over the course of years to diabetic retinopathy. Thevascular changes cause hypoperfusion of capillary regions. This leads tolipid deposits (hard exudates) and to vasoproliferation. The clinicalcourse is variable in patients with diabetes mellitus. In age-relateddiabetes (type II diabetes), capillary aneurysms appear first.Thereafter, because of the impaired capillary perfusion, hard and softexudates and dot-like hemorrhages in the retinal parenchyma appear. Inlater stages of diabetic retinopathy, the fatty deposits are arrangedlike a corona around the macula (retinitis circinata). These changes arefrequently accompanied by edema at the posterior pole of the eye. If theedema involves the macula there is an acute serious deterioration invision. The main problem in type I diabetes is the vascularproliferation in the region of the fundus of the eye. The standardtherapy is laser coagulation of the affected regions of the fundus ofthe eye. The laser coagulation is initially performed focally in theaffected areas of the retina. If the exudates persist, the area of lasercoagulation is extended. The center of the retina with the site ofsharpest vision, that is to say the macula and the papillomacularbundle, cannot be coagulated because the procedure would result indestruction of the parts of the retina which are most important forvision. If proliferation has already occurred, it is often necessary forthe foci to be very densely pressed on the basis of the proliferation.This entails destruction of areas of the retina. The result is acorresponding loss of visual field. In type I diabetes, lasercoagulation in good time is often the only chance of saving patientsfrom blindness.

In certain embodiments, the RPE cells of the invention may be used totreat disorders of the central nervous system. RPE cells may betransplanted into the CNS. To date, a number of different cell typeshave been employed in animal experiments or in patients with Parkinson'sdisease in clinical studies. Examples are fetal cells obtained frombrains of human fetuses. Fetal cells from the ventral midbrain ordopaminergic neurons have already been transplanted in clinical studieson more than 300 patients with Parkinson's disease (for review, seeAlexi T, Borlongan C V, Faull R L, Williams C E, Clark R G, Gluckman PD, Hughes P E (2000) (Neuroprotective strategies for basal gangliadegeneration: Parkinson's and Huntington's diseases. Prog Neurobiol 60:409 470). A number of different cell types, including non-neuronalcells, e.g. cells from the adrenal cortex, Sertoli cells on the gonadsor glomus cells from the carotid bodies, fibroblasts or astrocytes, havebeen used in patients with Parkinson's disease or in animal models withthe aim of replacing dopamine spontaneously or after gene transfer(Alexi et al. 2000, supra). The survival rate of transplanted fetaldopaminergic neurons is S 8%, which was enough to cause a slightimprovement in the signs and symptoms (Alexi ct al. 2000, supra).

In recent years, neuronal stem cells from brains of adult vertebrateshave been isolated, expanded in vitro and reimplanted into the CNS,after which they differentiated into pure neurons. Their function in theCNS remains uncertain, however. Neuronal precursor cells have also beenused for gene transfer (Raymon H K, Thode S, Zhou J, Friedman G C,Pardinas J R, Barrere C, Johnson R M, Sah D W (1999) Immortalized humandorsal root ganglion cells differentiate into neurons with nociceptiveproperties. J Neurosci 19: 5420 5428). Schwann cells which overexpressedNGF and GDNF had neuroprotective effects in models of Parkinsonism(Wilby M J, Sinclair S R, Muir E M, Zietlow R, Adcock K H, Horellou P,Rogers J H, Dunnett S B, Fawcett J W (1999) A glial cell line-derivedneurotrophic factor-secreting clone of the Schwann cell line SCTM41enhances survival and fiber outgrowth from embryonic nigral neuronsgrafted to the striatum and to the lesioned Substantia nigra. J Neurosci19: 2301 2312).

Another aspect of the present invention is therefore the use of pigmentepithelial cells for the therapy of nerve diseases, in particular adisease of the nervous system, preferably of the CNS, especially ofParkinson's disease.

An example of a common disease of the CNS is Parkinson's disease whichis a chronic degenerative disease of the brain. The disease is caused bydegeneration of specialized neuronal cells in the region of the basalganglia. The death of dopaminergic neurons results in reduced synthesisof dopamine, an important neurotransmitter, in patients with Parkinson'sdisease. The standard therapy is medical therapy with L-dopa. L-Dopa ismetabolized in the basal ganglia to dopamine and there takes over thefunction of the missing endogenous neurotransmitter. However, L-dopatherapy loses its activity after some years.

Animal models of retinitis pigmentosa that may be treated or used totest the efficacy of the RPE cells produced using the methods describedherein include rodents (rd mouse, RPE-65 knockout mouse, tubby-likemouse, RCS rat), cats (Abyssinian cat), and dogs (cone degeneration “cd”dog, progressive rod-cone degeneration “pred” dog, early retinaldegeneration “erd” dog, rod-cone dysplasia 1, 2 & 3 “rcd1, rcd2 & rcd3”dogs, photoreceptor dysplasia “pd” dog, and Briard “RPE-65” (dog)).

Another embodiment of the present invention is a method for thederivation of RPE lines or precursors to RPE cells that have anincreased ability to prevent neovascularization. Such cells can beproduced by aging a somatic cell from a patient such that telomerase isshortened where at least 10% of the normal replicative lifespan of thecell has been passed, then the use of said somatic cell as a nucleartransfer donor cell to create cells that overexpress angiogenesisinhibitors such as Pigment Epithelium Derived Factor (PEDF/EPC-1).Alternatively such cells may be genetically modified with exogenousgenes that inhibit neovascularization.

The invention contemplates that preparations of RPE cells differentiatedfrom human pluripotent stem cells (e.g., human embryonic stem cells, iPScells, or other pluripotent stem cells) can be used to treat any of theforegoing diseases or conditions, as well as injuries of the endogenousRPE layer. These diseases can be treated with preparations of RPE cellscomprising a mixture of differentiated RPE cells of varying levels ofmaturity, as well as with preparations of differentiated RPE cells thatare enriched for mature differentiated RPE cells or differentiated RPEcells.

Modes of Administration

RPE cells of the invention may be administered topically, systemically,or locally, such as by injection (e.g., intravitreal injection), or aspart of a device or implant (e.g., a sustained release implant). Forexample, the cells of the present invention may be transplanted into thesubretinal space by using vitrectomy surgery.

Depending on the method of administration, RPE cells can be added tobuffered and electrolyte balanced aqueous solutions, buffered andelectrolyte balanced aqueous solutions with a lubricating polymer,mineral oil or petrolatum-based ointment, other oils, liposomes,cylcodextrins, sustained release polymers or gels. These preparationscan be administered topically to the eye 1 to 6 times per day for aperiod up to the lifetime of the patient.

In certain embodiments, methods of treating a patient suffering from acondition associated with retinal degeneration comprise administering acomposition of the invention locally (e.g., by intraocular injection orinsertion of a sustained release device that releases a composition ofthe invention), by topical means or by systemic administration (e.g., byroutes of administration that allow in vivo systemic absorption oraccumulation of drugs in the blood stream followed by distributionthroughout the entire body, including, without limitation, byintravenous, subcutaneous, intraperitoneal, inhalation, oral,intrapulmonary and intramuscular routes). Intraocular administration ofcompositions of the invention includes, for example, delivery into thevitreous body, transcorneally, sub-conjunctival, juxtascleral, posteriorscleral, and sub-tenon portions of the eye. See, for example, U.S. Pat.Nos. 6,943,145; 6,943,153; and 6,945,971, the contents of which arehereby incorporated by reference.

RPE cells of the invention may be delivered in a pharmaceuticallyacceptable ophthalmic formulation by intraocular injection. Whenadministering the formulation by intravitreal injection, for example,the solution should be concentrated so that minimized volumes may bedelivered. Concentrations for injections may be at any amount that iseffective and non-toxic, depending upon the factors described herein. Insome embodiments, RPE cells for treatment of a patient are formulated atdoses of about 10⁴ cells/mL. In other embodiments, RPE cells fortreatment of a patient are formulated at doses of about 10⁵, 10⁶, 10⁷,10⁸, 10⁹, or 10¹⁰ cells/mL.

RPE cells may be formulated for delivery in a pharmaceuticallyacceptable ophthalmic vehicle, such that the composition is maintainedin contact with the ocular surface for a sufficient time period to allowthe cells to penetrate the affected regions of the eye, as for example,the anterior chamber, posterior chamber, vitreous body, aqueous humor,vitreous humor, cornea, iris/ciliary, lens, choroid, retina, sclera,suprachoridal space, conjunctiva, subconjunctival space, episcleralspace, intracorneal space, epicomeal space, pars plana,surgically-induced avascular regions, or the macula. Products andsystems, such as delivery vehicles, comprising the agents of theinvention, especially those formulated as pharmaceutical compositions—aswell as kits comprising such delivery vehicles and/or systems—are alsoenvisioned as being part of the present invention.

In certain embodiments, a therapeutic method of the invention includesthe step of administering RPE cells of the invention as an implant ordevice. In certain embodiments, the device is bioerodible implant fortreating a medical condition of the eye comprising an active agentdispersed within a biodegradable polymer matrix, wherein at least about75% of the particles of the active agent have a diameter of less thanabout 10 μm. The bioerodible implant is sized for implantation in anocular region. The ocular region can be any one or more of the anteriorchamber, the posterior chamber, the vitreous cavity, the choroid, thesuprachoroidal space, the conjunctiva, the subconjunctival space, theepiscleral space, the intracorneal space, the epicorneal space, thesclera, the pars plana, surgically-induced avascular regions, themacula, and the retina. The biodegradable polymer can be, for example, apoly(lactic-co-glycolic)acid (PLGA) copolymer. In certain embodiments,the ratio of lactic to glycolic acid monomers in the polymer is about25/75, 40/60, 50/50, 60/40, 75/25 weight percentage, more preferablyabout 50/50. Additionally, the PLGA copolymer can be about 20, 30, 40,50, 60, 70, 80 to about 90 percent by weight of the bioerodible implant.In certain preferred embodiments, the PLGA copolymer can be from about30 to about 50 percent by weight, preferably about 40 percent by weightof the bioerodible implant.

The volume of composition administered according to the methodsdescribed herein is also dependent on factors such as the mode ofadministration, number of RPE cells, age and weight of the patient, andtype and severity of the disease being treated. For example, ifadministered orally as a liquid, the liquid volume comprising acomposition of the invention may be from about 0.5 milliliters to about2.0 milliliters, from about 2.0 milliliters to about 5.0 milliliters,from about 5.0 milliliters to about 10.0 milliliters, or from about 10.0milliliters to about 50.0 milliliters. If administered by injection, theliquid volume comprising a composition of the invention may be fromabout 5.0 microliters to about 50 microliters, from about 50 microlitersto about 250 microliters, from about 250 microliters to about 1milliliter, from about 1 milliliter to about 5 milliliters, from about 5milliliters to about 25 milliliters, from about 25 milliliters to about100 milliliters, or from about 100 milliliters to about 1 liter.

If administered by intraocular injection, RPE cells can be delivered oneor more times periodically throughout the life of a patient. For exampleRPE cells can be delivered once per year, once every 6-12 months, onceevery 3-6 months, once every 1-3 months, or once every 1-4 weeks.Alternatively, more frequent administration may be desirable for certainconditions or disorders. If administered by an implant or device, RPEcells can be administered one time, or one or more times periodicallythroughout the lifetime of the patient, as necessary for the particularpatient and disorder or condition being treated. Similarly contemplatedis a therapeutic regimen that changes over time. For example, morefrequent treatment may be needed at the outset (e.g., daily or weeklytreatment). Over time, as the patient's condition improves, lessfrequent treatment or even no further treatment may be needed.

In certain embodiments, patients are also administered immunosuppressivetherapy, either before, concurrently with, or after administration ofthe RPE cells.

Immunosuppressive therapy may be necessary throughout the life of thepatient, or for a shorter period of time.

In certain embodiments, RPE cells of the present invention areformulated with a pharmaceutically acceptable carrier. For example, RPEcells may be administered alone or as a component of a pharmaceuticalformulation. The subject compounds may be formulated for administrationin any convenient way for use in human medicine. In certain embodiments,pharmaceutical compositions suitable for parenteral administration maycomprise the RPE cells, in combination with one or more pharmaceuticallyacceptable sterile isotonic aqueous or nonaqueous solutions,dispersions, suspensions or emulsions, or sterile powders which may bereconstituted into sterile injectable solutions or dispersions justprior to use, which may contain antioxidants, buffers, bacteriostats,solutes which render the formulation isotonic with the blood of theintended recipient or suspending or thickening agents. Examples ofsuitable aqueous and nonaqueous carriers which may be employed in thepharmaceutical compositions of the invention include water, ethanol,polyols (such as glycerol, propylene glycol, polyethylene glycol, andthe like), and suitable mixtures thereof, vegetable oils, such as oliveoil, and injectable organic esters, such as ethyl oleate. Properfluidity can be maintained, for example, by the use of coatingmaterials, such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants.

The compositions of the invention may also contain adjuvants, such aspreservatives, wetting agents, emulsifying agents and dispersing agents.Prevention of the action of microorganisms may be ensured by theinclusion of various antibacterial and antifungal agents, for example,paraben, chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like in the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof one or more agents that delay absorption, such as, e.g., aluminummonostearate and gelatin.

When administered, the therapeutic composition for use in this inventionis, of course, in a pyrogen-free, physiologically acceptable form.Further, the composition may desirably be encapsulated or injected in aviscous form into the vitreous humor for delivery to the site of retinalor choroidal damage.

Engineering MHC Genes in Human Embryonic Stem Cells to ObtainReduced-Complexity RPE Cells

The human embryonic stem cells used as the starting point for the methodof producing RPE cells of this invention may also be derived from alibrary of human embryonic stem cells, each of which is hemizygous orhomozygous for at least one MHC allele present in a human population. Incertain embodiments, each member of said library of stem cells ishemizygous or homozygous for a different set of MHC alleles relative tothe remaining members of the library. In certain embodiments, thelibrary of stem cells is hemizygous or homozygous for all MHC allelesthat are present in a human population. In the context of thisinvention, stem cells that are homozygous for one or morehistocompatibility antigen genes include cells that are nullizygous forone or more (and in some embodiments, all) such genes. Nullizygous for agenetic locus means that the gene is null at that locus, i.e., bothalleles of that gene are deleted or inactivated. Stem cells that arenullizygous for all MHC genes may be produced by standard methods knownin the art, such as, for example, gene targeting and/or loss ofheterozygosity (LOH). See, for example, United States patentpublications US 20040091936, US 20030217374 and US 20030232430, and U.S.provisional application No. 60/729,173, the disclosures of all of whichare hereby incorporated by reference herein.

Accordingly, the present invention relates to methods of obtaining RPEcells, including a library of RPE cells, with reduced MHC complexity.RPE cells with reduced MHC complexity will increase the supply ofavailable cells for therapeutic applications as it will eliminate thedifficulties associated with patient matching. Such cells may be derivedfrom stem cells that are engineered to be hemizygous or homozygous forgenes of the MHC complex.

A human ES cell may comprise modifications to one of the alleles ofsister chromosomes in the cell's MHC complex. A variety of methods forgenerating gene modifications, such as gene targeting, may be used tomodify the genes in the MHC complex. Further, the modified alleles ofthe MHC complex in the cells may be subsequently engineered to behomozygous so that identical alleles are present on sister chromosomes.Methods such as loss of heterozygosity (LOH) may be utilized to engineercells to have homozygous alleles in the MHC complex. For example, one ormore genes in a set of MHC genes from a parental allele can be targetedto generate hemizygous cells. The other set of MHC genes can be removedby gene targeting or LOH to make a null line. This null line can be usedfurther as the embryonic cell line in which to drop arrays of the HLAgenes, or individual genes, to make a hemizygous or homozygous bank withan otherwise uniform genetic background.

In one aspect, a library of ES cell lines, wherein each member of thelibrary is homozygous for at least one HLA gene, is used to derive RPEcells according to the methods of the present invention. In anotheraspect, the invention provides a library of RPE cells (and/or RPElineage cells), wherein several lines of ES cells are selected anddifferentiated into RPE cells. These RPE cells and/or RPE lineage cellsmay be used for a patient in need of a cell-based therapy.

Accordingly, certain embodiments of this invention pertain to a methodof administering human RPE cells that have been derived fromreduced-complexity embryonic stem cells to a patient in need thereof. Incertain embodiments, this method comprises the steps of: (a) identifyinga patient that needs treatment involving administering human RPE cellsto him or her; (b) identifying MHC proteins expressed on the surface ofthe patient's cells; (c) providing a library of human RPE cells ofreduced MHC complexity made by the method for producing RPE cells of thepresent invention; (d) selecting the RPE cells from the library thatmatch this patient's MHC proteins on his or her cells; (e) administeringany of the cells from step (d) to said patient. This method may beperformed in a regional center, such as, for example, a hospital, aclinic, a physician's office, and other health care facilities. Further,the RPE cells selected as a match for the patient, if stored in smallcell numbers, may be expanded prior to patient treatment.

Other Commercial Applications and Methods

Certain aspects of the present invention pertain to the production ofRPE cells to reach commercial quantities. In particular embodiments, RPEcells are produced on a large scale, stored if necessary, and suppliedto hospitals, clinicians or other healthcare facilities. Once a patientpresents with an indication such as, for example, Stargardt's maculardystrophy, age related macular degeneration, or retinitis pigmentosa,RPE cells can be ordered and provided in a timely manner. Accordingly,the present invention relates to methods of producing RPE cells toattain cells on a commercial scale, cell preparations comprising RPEcells derived from said methods, as well as methods of providing (i.e.,producing, optionally storing, and selling) RPE cells to hospitals andclinicians.

Accordingly certain aspects of the present invention relate to methodsof production, storage, and distribution of RPE cells produced by themethods disclosed herein. Following RPE production, RPE cells may beharvested, purified and optionally stored prior to a patient'streatment. RPE cells may optionally be patient specific or specificallyselected based on HLA or other immunologic profile.

Thus in particular embodiments, the present invention provides methodsof supplying RPE cells to hospitals, healthcare centers, and clinicians,whereby RPE cells produced by the methods disclosed herein are stored,ordered on demand by a hospital, healthcare center, or clinician, andadministered to a patient in need of RPE cell therapy. In alternativeembodiments, a hospital, healthcare center, or clinician orders RPEcells based on patient specific data, RPE cells are produced accordingto the patient's specifications and subsequently supplied to thehospital or clinician placing the order.

In certain embodiments, the method of differentiating RPE cells fromhuman embryonic stem cells is conducted in accordance with GoodManufacturing Practices (GMP). In certain embodiments, the initialderivation or production of human embryonic stem cells is also conductedin accordance with Good Manufacturing Practices (GMP). The cells may betested at one or more points throughout the differentiation protocol toensure, for example, that there is no viral, bacterial, or fungalinfection or contamination in the cells or culture medium. Similarly,the human embryonic stem cells used as starting material may be testedto ensure that there is no viral, bacterial, or fungal infection orcontamination.

In certain embodiments, the production of differentiated RPE cells ormature differentiated RPE cells is scaled up for commercial use. Forexample, the method can be used to produce at least 1×10⁵, 5×10⁵, 1×10⁶,5×10⁶, 1×10⁷, 5×10⁷, 1×10⁸, 5×10⁸, 1×10⁹, 5×10⁹, or 1×10¹⁰ RPE cells.

Further aspects of the invention relate to a library of RPE cells thatcan provide matched cells to potential patient recipients. Accordingly,in one embodiment, the invention provides a method of conducting apharmaceutical business, comprising the step of providing RPE cellpreparations that are homozygous for at least one histocompatibilityantigen, wherein cells are chosen from a bank of such cells comprising alibrary of RPE cells that can be expanded by the methods disclosedherein, wherein each RPE cell preparation is hemizygous or homozygousfor at least one MHC allele present in the human population, and whereinsaid bank of RPE cells comprises cells that are each hemizygous orhomozygous for a different set of MHC alleles relative to the othermembers in the bank of cells. As mentioned above, gene targeting or lossof heterozygosity may be used to generate the hemizygous or homozygousMHC allele stem cells used to derive the RPE cells. In one embodiment,after a particular RPE cell preparation is chosen to be suitable for apatient, it is thereafter expanded to reach appropriate quantities forpatient treatment. Methods of conducting a pharmaceutical business mayalso comprise establishing a distribution system for distributing thepreparation for sale or may include establishing a sales group formarketing the pharmaceutical preparation.

Other aspects of the invention relate to the use of the RPE cells of thepresent invention as a research tool in settings such as apharmaceutical, chemical, or biotechnology company, a hospital, or anacademic or research institution. Such uses include the use of RPE cellsdifferentiated from embryonic stem cells in screening assays toidentify, for example, agents that can be used to promote RPE survivalin vitro or in vivo, or that can be used to promote RPE maturation.Identified agents can be studied in vitro or in animal models toevaluate, for example, their potential use alone or in combination withRPE cells.

The present invention also includes methods of obtaining human ES cellsfrom a patient and then generating and expanding RPE cells derived fromthe ES cells. These RPE cells may be stored. In addition, these RPEcells may be used to treat the patient from which the ES were obtainedor a relative of that patient.

As the methods and applications described above relate to treatments,pharmaceutical preparations, and the storing of RPE cells, the presentinvention also relates to solutions of RPE cells that are suitable forsuch applications. The present invention accordingly relates tosolutions of RPE cells that are suitable for injection into a patient.Such solutions may comprise cells formulated in a physiologicallyacceptable liquid (e.g., normal saline, buffered saline, or a balancedsalt solution). The number of cells in the solution may be at leastabout 10² and less than about 10⁹ cells. In other embodiments, thenumber of cells in the solution may range from about 10¹, 10², 5×10²,10³, 5×10³, 10⁴, 10⁵, 10⁶, 10⁷, or 10⁸ to about 5×10², 10³, 5×10³, 10⁴,10⁵, 10⁶, 10⁷, 10⁸, or 10⁹, where the upper and lower limits areselected independently, except that the lower limit is always less thanthe upper limit. Further, the cells may be administered in a single orin multiple administrations.

Cells provided by the methods described herein may be used immediatelyor may be frozen and cryopreserved for days or years. Thus, in oneembodiment, the present invention provides a cryopreserved preparationof RPE cells, wherein said cryopreserved preparation comprises at leastabout 10¹, 10², 5×10², 10³, 5×10³, 10⁴, 5×10⁴, 10⁵, 5×10⁵, or 10⁶.Cryopreserved preparations may further comprise at least about 5×10⁶,10⁷, 5×10⁷, 10⁸, 15×0⁸, 10⁹, 5×10⁹, or 10¹⁰ cells. Similarly providedare methods of cryopreserving RPE cells. RPE cells may be cryopreservedimmediately following differentiation, following in vitro maturation, orafter some period of time in culture. The RPE cells in the preparationsmay comprise a mixture of differentiated RPE cells and mature RPE cells.

Other Pluripotent Cells

The foregoing discussion focuses on the use of human embryonic stemcells as the starting material for making unique RPE cells, as well aspreparations and methods of using RPE cells differentiated from humanembryonic stem cells. However, the methods and uses detailed above cansimilarly be used to generate RPE cells (and suitable preparations)using other types of human pluripotent stem cells as starting material.Accordingly, the invention contemplates that any of the foregoing orfollowing aspects and embodiments of the invention can be similarlyapplied to methods and uses of RPE cells differentiated from other typesof human pluripotent stem cells. Of particular note, given that inducedpluripotent stem (iPS) cells have the characteristics of embryonic stemcells, such cells can be used to produce RPE cells that are identical orsubstantially identical to RPE cells differentiated from embryonic stemcells.

As used herein, the term “pluripotent stem cells” includes embryonicstem cells, embryo-derived stem cells, and induced pluripotent stemcells, regardless of the method by which the pluripotent stem cells arederived. Pluripotent stem cells are defined functionally as stem cellsthat: (a) are capable of inducing teratomas when transplanted inimmunodeficient (SCID) mice; (b) are capable of differentiating to celltypes of all three germ layers (e.g., can differentiate to ectodermal,mesodermal, and endodermal cell types); and (c) express one or moremarkers of embryonic stem cells (e.g., express Oct 4, alkalinephosphatase, SSEA-3 surface antigen, SSEA-4 surface antigen, nanog,TRA-1-60, TRA-1-81, SOX2, REX1, etc). Exemplary pluripotent stem cellscan be generated using, for example, methods known in the art. Exemplarypluripotent stem cells include embryonic stem cells derived from the ICMof blastocyst stage embryos, as well as embryonic stem cells derivedfrom one or more blastomeres of a cleavage stage or morula stage embryo(optionally without destroying the remainder of the embryo). Suchembryonic stem cells can be generated from embryonic material producedby fertilization or by asexual means, including somatic cell nucleartransfer (SCNT), parthenogenesis, cellular reprogramming, andandrogenesis. Further exemplary pluripotent stem cells include inducedpluripotent stem cells (iPS cells) generated by reprogramming a somaticcell by expressing or inducing the expression of a combination offactors (herein referred to as reprogramming factors). iPS cells can begenerated using fetal, postnatal, newborn, juvenile, or adult somaticcells. In certain embodiments, factors that can be used to reprogramsomatic cells to pluripotent stem cells include, for example, acombination of Oct4 (sometimes referred to as Oct 3/4), Sox2, c-Myc, andKlf4. In other embodiments, factors that can be used to reprogramsomatic cells to pluripotent stem cells include, for example, acombination of Oct 4, Sox2, Nanog, and Lin28. In other embodiments,somatic cells are reprogrammed by expressing at least 2 reprogrammingfactors, at least three reprogramming factors, or four reprogrammingfactors. In other embodiments, additional reprogramming factors areidentified and used alone or in combination with one or more knownreprogramming factors to reprogram a somatic cell to a pluripotent stemcell.

Embryonic stem cells are one example of pluripotent stem cells. Anotherexample are induced pluripotent stem (iPS) cells.

In certain embodiments, the pluripotent stem cell is an embryonic stemcell or embryo-derived cell. In other embodiments, the pluripotent stemcell is an induced pluripotent stem cell. In certain embodiments, thepluripotent stem cell is an induced pluripotent stem cell produced byexpressing or inducing the expression of one or more reprogrammingfactors in a somatic cell. In certain embodiments, the somatic cell is afibroblast, such as a dermal fibroblast, synovial fibroblast, or lungfibroblast. In other embodiments, the somatic cell is not a fibroblast,but rather is a non-fibroblastic somatic cell. In certain embodiments,the somatic cell is reprogrammed by expressing at least tworeprogramming factors, at least three reprogramming factors, or fourreprogramming factors. In other embodiments, the somatic cell isreprogrammed by expressing at least four, at least five, or at least sixreprogramming factors. In certain embodiments, the reprogramming factorsare selected from Oct 3/4, Sox2, Nanog, Lin28, c-Myc, and KIf4. In otherembodiments, the set of reprogramming factors expressed includes atleast one, at least two, at least three, or at least four of theforegoing list of reprogramming factors, and optionally includes one ormore other reprogramming factors. In certain embodiments, expression ofat least one, at least two, at least three, or at least four of theforegoing or other reprogramming factors is induced by contacting thesomatic cells with one or more agents, such as a small organic moleculeagents, that induce expression of one or more reprogramming factors. Incertain embodiments, the somatic cell is reprogramming using acombinatorial approach wherein one or more reprogramming factor isexpressed (e.g., using a viral vector, plasmid, and the like) and theexpression of one or more reprogramming factor is induced (e.g., using asmall organic molecule.).

In certain embodiments, reprogramming factors are expressed in thesomatic cell by infection using a viral vector, such as a retroviralvector or a lentiviral vector. In other embodiments, reprogrammingfactors are expressed in the somatic cell using a non-integrativevector, such as an episomal plasmid. When reprogramming factors areexpressed using non-integrative vectors, the factors can be expressed inthe cells using electroporation, transfection, or transformation of thesomatic cells with the vectors.

In certain embodiments, the pluripotent stem cells are generated fromsomatic cells, and the somatic cells are selected from embryonic, fetal,neonatal, juvenile, or adult cells.

Methods for making iPS cells by expressing or inducing the expression ofreprogramming factors are known in the art. Briefly, somatic cells areinfected, transfected, or otherwise transduced with expression vectorsexpressing reprogramming factors. In the case of mouse, expression offour factors (Oct3/4, Sox2, c-myc, and Klf4) using integrative viralvectors was sufficient to reprogram a somatic cell. In the case ofhumans, expression of four factors (Oct3/4, Sox2, Nanog, and Lin28)using integrative viral vectors was sufficient to reprogram a somaticcell. However, expression (or induction of expression) of fewer factorsor other reprogramming factors may also be sufficient. Additionally, theuse of integrative vectors is only one mechanism for expressingreprogramming factors in the cells. Other methods including, forexample, the use of non-integrative vectors can be used.

In certain embodiments, expression of at least one, at least two, atleast three, or at least four of the foregoing or other reprogrammingfactors is induced by contacting the somatic cells with one or moreagents, such as a small organic molecule agents, that induce expressionof one or more reprogramming factors. In certain embodiments, thesomatic cell is reprogramming using a combinatorial approach wherein oneor more reprogramming factor is expressed (e.g., using a viral vector,plasmid, and the like) and the expression of one or more reprogrammingfactor is induced (e.g., using a small organic molecule.).

Once the reprogramming factors are expressed in the cells, the cells arecultured. Over time, cells with ES characteristics appear in the culturedish. The cells can be picked and subcultured based on, for example, ESmorphology, or based on expression of a selectable or detectable marker.The cells are cultured to produce a culture of cells that look like EScells. These cells are putative iPS cells.

To confirm the pluripotency of the iPS cells, the cells can be tested inone or more assays of pluripotency. For examples, the cells can betested for expression of ES cell markers; the cells can be evaluated forability to produce teratomas when transplanted into SCID mice; the cellscan be evaluated for ability to differentiate to produce cell types ofall three germ layers.

Once pluripotent iPS cells are obtained (either freshly derived or froma bank or stock of previously derived cells), such cells can be used tomake RPE cells.

In certain embodiments, the making of iPS cells is an initial step inthe production of RPE cells. In other embodiments, previously derivediPS cells are used. In certain embodiments, iPS cells are specificallygenerated using material from a particular patient or matched donor withthe goal of generating tissue-matched RPE cells. In certain embodiments,the iPS cells are universal donor cells that are not substantiallyimmunogenic.

The present invention will now be more fully described with reference tothe following examples, which are illustrative only and should not beconsidered as limiting the invention described above.

EXAMPLES

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

The pluripotency of embryonic stem cells is maintained in-part by thedelicate reciprocal balance of the two transcription factors Oct4(Pou5f1) and Nanog. During ES cell differentiation, the expression ofthese genes is downregulated, and recent evidence has suggestedhypermethylation of the genes encoding these proteins to be responsible.Loss of the expression of either or both of these genes results intranscriptional activation of genes associated with cellulardifferentiation.

The retinal pigmented epithelium (RPE) develops from the neuroectodermand is located adjacent to the neural retina and choroid, providing abarrier between the vascular system and the retina. The data providedherein indicates that RPE cells are genetically and functionallydistinguished from surrounding photoreceptors after terminaldifferentiation, although the cells may share a common progenitor.

This model indicates that elements unique to our culture method claimsact through FGF, EGF, WNT4, TGF-beta, and/or oxidative stress to signalMAP-Kinase and potential C-Jun terminal Kinase pathways to induce theexpression of the Paired-box 6 (PAX6) transcription factor. PAX6 actssynergistically with PAX2 to terminally differentiate mature RPE via thecoordination of Mit-F and Otx2 to transcribe RPE-specific genes such asTyrosinase (Tyr), and downstream targets such as RPE-65, Bestrophin,CRALBP, and PEDF.

In order to characterize developmental stages during the human embryonicstem cell (hESc) differentiation process into retinal pigmentedepithelium (RPE), several assays were used to identify the expressionlevels of genes key to each representative stage of development. It wasdiscovered that several genes were uniquely expressed as mRNA andprotein in RPE cells. For instance, it was discovered that PAX6 actswith PAX2 to terminally differentiate mature RPE cells via coordinationof Mit-F and Otx2 to transcribe RPE-specific genes such as Tyrosinase(Tyr), and downstream targets such as RPE-65, Bestrophin, CRALBP, andPEDF. Importantly, the RPE-specific signature of mRNA and proteinexpression was not only unique from hES cells, but also from fetal RPEand ARPE-19 cells. The RPE cells described herein expressed multiplegenes that were not expressed in hES cells, fetal RPE cells, or ARPE-19cells (FIGS. 3, 4, and 6). The unique expression of mRNA and proteins inthe RPE cells of the invention constitutes a set of markers that makethese RPE cells distinct from cells in the art, such as hES cells,ARPE-19 cells, and fetal RPE cells.

Example 1: RPE Differentiation and Culture

Cryopreserved hES cells were thawed and placed into suspension cultureon Lo-bind Nunclon Petri dishes in MDBK-Growth Medium (Sigma—SAFCBiosciences) or OptimPro SFM (Invitrogen) supplemented with L-Glutamine,Penicillin/Streptomycin, and B-27 supplement. The hES cells had beenpreviously derived from single blastomeres biopsied from early cleavagestage human embryos. The remainder of the human embryo was notdestroyed. Two hES cell line derived from single blastomeres wereused—MA01 and MA09. The cells were cultured for 7-14 days as embryoidbodies (EBs).

After 7-14 days, the EBs were plated onto tissue culture plates coatedwith gelatin from porcine skin. The EBs were grown as adherent culturesfor an additional 14-28 days in MDBK-Growth Medium or OptimPro SFMsupplemented with L-Glutamine, and Penicillin/Streptomycin, without B-27supplement.

From amongst the cells in the adherent culture of EBs, RPE cells becomevisible and are recognized by their cobblestone cellular morphology andemergence of pigmentation.

Example 2: RPE Isolation and Propagation

As differentiated RPE cells continue to appear in the adherent cultures,clusters of differentiated RPEs become visibly noticeable based on cellshape. Frozen collagenase IV (20 mg/ml) was thawed and diluted to 7mg/ml. The collagenase IV was applied to the adherent culture containingRPE clusters (1.0 ml to each well in a 6-well plate). Over approximately1-3 hours, the collagenase IV dissociated the cell clusters. Bydissociating the RPE clusters from other cells in the culture, anenriched suspension of RPE cells was obtained. The enriched RPE cellsuspension was removed from the culture plate and transferred to a 100mm. tissue culture dish with 10 ml of MEF medium. Pigmented clumps aretransferred with a stem cell cutting tool (Swemed-Vitrolife) to a wellof a 6-well plate containing 3 ml of MEF media. After all clumps havebeen picked up, the suspension of pigmented cells is transferred to a 15ml conical tube containing 7 ml of MEF medium and centrifuged at 1000rpm for five minutes. The supernatant is removed. 5 ml of a 1:1 mixtureof 0.25% trypsin and cell dissociation buffer is added to the cells. Thecells are incubated for 10 minutes at 37° C. The cells are dispersed bypipetting in a 5 ml pipette until few clumps are remaining. 5 ml of MEFmedium is added to the cells and the cells centrifuged at 1000 rpm for 5minutes. The supernatant is removed and the cells are plated on gelatincoated plates with a split of 1:3 of the original culture in EGM-2culture medium (Cambrex).

The culture of RPE cells was expanded by continued culture in EGM-2medium. The cells were passaged, as necessary, at a 1:3 to 1:6 ratiousing a 1:1 mixture of 0.25% trypsin EDTA and Cell Dissociation Buffer.

To enrich for mature differentiated RPE cells, the cells were grown tonear confluence in EGM-2. The medium was then changed to MDBK-MM (SAFCBiosciences) to help further promote maturation of the RPE cells.

Example 3: RPE-Specific mRNA Expression Measured by Quantitative,Real-Time, Reverse Transcription PCR (qPCR)

In order to characterize developmental stages during the human embryonicstem cell (hES) differentiation process into retinal pigmentedepithelium (RPE) several assays have been employed to identify theexpression levels of genes key to each representative stage ofdevelopment. qPCR was developed to provide a quantitative and relativemeasurement of the abundance of cell type-specific mRNA transcripts ofinterest in the RPE differentiation process. qPCR was used to determinegenes that are uniquely expressed in human embryonic stem cells, inneuroretinal cells during eye development, and in RPE cellsdifferentiated from human embryonic stem cells. The genes for each celltype are listed below in Table 1.

TABLE 1 Genes specific to hES, neuroretina/eye, and hRPE cellshESc-Specific Neuroectoderm/Neuroretina RPE-Specific Genes Oct-4(POU5F1) CHX10 PAX-6 Nanog NCAM PAX-2 Rex-1 Nestin RPE-65 TDGF-1Beta-Tubulin PEDF SOX-2 CRAMP DPPA-2 Bestrophin MitF Otx-2 Tyr

It was determined that hES-specific genes included Oct-4 (POU5F1),Nanog, Rex-1, TDGF-1, SOX-2, and DPPA-2. Genes specific to neuralectoderm/neural retina include CHX10, NCAM, Nestin, and Beta-Tubulin. Bycontrast, RPE cells differentiated from human embryonic stem cells werefound to uniquely express PAX-6, PAX-2, RPE-65, PEDF, CRALBP,Bestrophin, MitF, Otx-2, and Tyr by qPCR measurement.

As evident from the qPCR data, hES-specific genes are grosslydownregulated (near 1000-fold) in RPE cells derived from hES, whereasgenes specific for RPE and neuroectoderm are vastly upregulated(approximately 100-fold) in RPE cells derived from hES.

In addition, qPCR analysis of fully mature RPE demonstrated a high levelexpression of the RPE-specific markers RPE65, Tyrosinase, PEDF,Bestrophin, MitF, and Pax6. This finding further elaborates on theontogeny depicted above and agrees with current literature regarding thePax2-induced regulation of MitF and downstream activation of genesassociated with terminally differentiated RPE.

Example 4: RPE-Specific Protein Expression Identified by Western BlotAnalysis

In order to validate the qPCR results above, and to identify proteinsuniquely expressed in RPE cells, a subset of hES-specific andRPE-specific markers were chosen as candidates to assay by western blot,thereby demonstrating translation of the message detected by PCR.Western analysis provides an absolute measure of the robustness of otherassays with semi-quantitative (via densitometry) and qualitative data.Results are pictured in FIG. 6. Actin was used as protein loadingcontrol.

RPE cells derived from hES cells did not express the hES-specificproteins Oct-4, Nanog, and Rex-1, whereas they expressed RPE65, CRALBP,PEDF, Bestrophin, PAX6, and Otx2. These proteins are therefore prominentmarkers of RPE cells differentiated from hES cells. By contrast, APRE-19cells showed an inconclusive pattern of proteomic marker expression.

Example 5: Microarray Gene Expression Profiling of RPE Cells

Manually-purified, hES cell-differentiated hRPE in vitro undergosignificant morphological events in culture during the expansion phase.Single-cell suspensions plated in thin cultures depigment and cellsincrease in surface area. hRPE cells maintain this morphology duringexpansion when the cells are rapidly dividing. However, when celldensity reaches maximal capacity, RPE take on their characteristicphenotypic hexagonal shape and increase pigmentation level byaccumulating melanin and lipofuscin.

The level of pigmentation played a major role in our pharmacology studyin the RCS rat model. Therefore, we performed global gene expressionanalysis via microarray on hRPE cells derived from both of the singleblastomere-derived hES cell lines MA01 and MA09. Additionally, fetalRPE, ARPE-19, and retinoblastoma cell lines were analyzed as controls.

Our data indicates that this phenotypic change is driven by a change inthe global gene expression pattern of these cells, specifically withregard to the expression of PAX6, PAX2, Otx2, MitF, and Tyr.

FIG. 7 depicts a principle components analysis plot scattering of eachsample based upon the minimal number of genes accounting for variabilityamongst each sample. Component 1, representing 69% of the variabilityrepresents the cell type, whereas Component 2, represents the cell line(i.e., genetic variability). As can clearly be seen, a near-linearscatter of gene expression profiles characterizes the developmentalontogeny of hRPE derived from hES cells.

Based on ANOVA analysis comparing the respective hES cell line to itsRPE counterpart, we selected the 100 highest and lowest expressed genes,and performed computational analysis to select genes related topleuripotency and eye development. Upregulated genes are shown in Table2. Down regulated genes are shown in Table 3.

TABLE 2 Upregulated genes of interest reported on microarrays GeneAssociated Symbol Gene Name with Description BEST1/VMD2 bestrophin RPEPredominantly expressed in the basolateral membrane of (vitelliformdevelopment the retinal pigment epithelium. Forms calcium-sensitivemacular chloride channels. May conduct other physiologically dystrophy2) significant anions such as bicarbonate. Defects in BEST 1 are thecause of vitelliform macular dystrophy type 2 (VMD2); also known as Bestmacular dystrophy (BMD). VMD2 is an autosomal dominant form of maculardegeneration that usually begins in childhood or adolescence. VMD2 ischaracterized by typical “egg yolk” macular lesions due to abnormalaccumulation of lipofuscin within and beneath the retinal pigmentedepithelium cells. Progression of the disease leads to destruction of theretinal pigmented epithelium and vision loss. Defects in BEST1 are acause of adult-onset vitelliform macular dystrophy (AVMD). AVMD is arare autosomal dominant disorder with incomplete penetrance and highlyvariable expression. Patients usually become symptomatic in the fourthor fifth decade of life with a protracted disease of decreased visualacuity. CLUL1(retinal) clusterin-like 1 retinal Associated strongly withcone photoreceptors and development appears in different tissuesthroughout retinal development. CRX cone-rod retinal Phosphoreceptor(cone, rod) specific paired-like homeo homeobox development domainprotein,expressed in developing and mature phosphoreeeptor cells,bindingand transactivating rhodopsin, homolog to Drosophila orthodentiele(Otx). Essential for the maintenance of mammalian photoreceptors. CRYAAcrystailin, eye Crystallins are the dominant structural components ofthe alpha A development vertebrate eye lens. May contribute to thetransparency and refractive index of the lens. Defects in CRYAA are thecause of zonular central nuclear cataract one of a considerable numberof phenotypically and genotypically distinct forms of autosomal dominantcataract. This congenital cataract is a common major abnormality of theeye that frequently causes blindness in infants. Crystallins do not turnover as the lens ages, providing ample opportunity forpost-translational modificanons or oxidations. These modifications maychange crystallin solubility properties and favor senile cataract.CRYBA1 crystallin, beta eye Crystallins are the dominant structuralcomponents of the A1 development vertebrate eye lens. Crystallins do notturn over as the lens ages, providing ample opportunity for post-translational modifications or oxidations. These modifications maychange crystallin solubility properties and favor senile cataract.CRYBA2 crystallin, beta eye Crystallins are the dominant structuralcomponents of the A2 development vertebrate eye lens. Crystallins do notturn over as the lens ages, providing ample opportunity for post-translational modifications or oxidations. These modifications maychange crystallin solubility properties and favor senile cataract.CRYBA4 crystallin, beta eye Crystallins are the dominant structuralcomponents of the A4 development vertebrate eye lens. Defects in CRYBA4are the cause of lamellar cataract 2. Cataracts are a leading cause ofblindness worldwide, affecting all societies. A significant proportionof cases are genetically determined. More than 15 genes for cataractshave been identified, of which the crystallin genes are the mostcommonly mutated. Lamellar cataract 2 is an autosomal dominantcongenital cataract. Defects in CRYBA4 are a cause of isolatedmicrophthalmia with cataract 4 (MCOPCT4). Microphtalmia consists of adevelopment defect causing moderate or severe reduction in size of theeye. Opacities of the cornea and lens, searing of the retina andchoroid, and other abnormalities like cataract may also be presentCrystallins do not turn over as the lens ages, providing ampleopportunity for post-translational modifications or oxidations. Thesemodifications may change crystallin solubility properties and favorsenile cataract. CRYBB1 crystatlin, beta eye Crystallins are thedominant structural components of the B1 development vertebrate eyelens. CRYBB2 crystallin, beta eye Crystallins are the dominantstructural components of the B2 development vertebrate eye lens. Defectsin CRYBB2 are the cause of congenital cerulean cataract 2 (CCA2); alsoknown as congenital cataract blue dot type 2. CCA2 is a form ofautosomal dominant congenital cataract (ADCC). Cerulean cataracts haveperipheral bluish and white opacifications in concentric layers withoccasional central lesions arranged radially. Although the opacities maybe observed during fetal development and childhood, usually visualacuity is only mildly reduced until adulthood, when lens extraction isgenerally necessary. Defects in CRYBB2 are the cause of sutural cataractwith punctate and cerulean opacities (CSPC). The phenotype associatedwith this form of autosomal dominant congenital cataract differed fromall other forms of cataract reported. Defects in CRYBB2 are a cause ofCoppock-like cataract (CCL). Crystallins do not turn over as the lensages, providing ample opportunity for post-translational modificationsor oxidations. CRYBB3 crystallin, beta eye Crystallins are the dominantstructural components of the B3 development vertebrate eye lens. Defectsin CRYBB3 arc the cause of autosomal recessive congenital nuclearcataract 2(CATCN2); a form of nonsyndromic congenital cataract.Non-syndromic congenital cataracts vary markedly in severity andmorphology, affecting the nuclear, cortical, polar, or subcapsular partsof the lens or, in severe cases, the entire lens, with a variety oftypes of opacity. They are one of the major causes of vision loss inchildren worldwide and are responsible for approximately one third ofblindness in infants. Congenital cataracts can lead to permanentblindness by interfering with the sham focus of light on the retinaduring critical developmental intervals. Crystallins do not turn over asthe lens ages, providing ample opportunity for post-translationalmodifications or oxidations. These modifications may change crystallinsolubility properties and favor senile cataract. DCT/TYRP2 dopachromepigmented Tyrosine metabolism and Melanin biosynthesis. tautomerasecells (dopachrome delta- isomerase, tyrosine- related protein 2) LHX2LIM development/ Transcriptional regulatory protein involved in thecontrol homeobox 2 differentiation of cell differentiation in developinglymphoid and neural cell types. LIM2 lens intrinsic eye Present in thethicker 16-17 nm junctions of mammalian membrane development lens fibercells, where it may contribute to cell junctional protein 2,organization. Acts as a receptor for calmodulin. May play 19 kDa animportant role in both lens development and cataractogenesis. MITFmicrophihalrni RPE Transcription factor for tyrosinase and tyrosinase-related a-associated development protein 1. Binds to a symmetrical DNAsequence (E- transcription boxes) (5′-CACGTG-3′) found in the tyrosinasefactor promoter. Plays a critical role in the differentiation of variouscell types as neural crest- derived melanocytes, mast cells, osteoclastsand optic cup-derived retinal pigmented epithelium. Highly expressed inretinal pigmented epithelium OCA2 oculocutaneou pigmented Could beinvolved in the transport of tyrosine, the s albinism II cells precursorto melanin synthesis, within the melanocyte. (pink-eye Regulates the pHof melanosome and the melanosome dilution maturation. One of thecomponents of the mammalian homolog, pigmentary system. Seems toregulate the mouse) postranslational processing of tyrosinase, whichcatalyzes the limiting reaction in melanin synthesis. May serve as a keycontrol point at which ethnic skin color variation is determined. Majordeterminant of brown and/or blue eye color. Defects in OCA2 are thecause of oculocutaneous albinism type II (OCA2). OCA2 is an autosomalrecessive form of albinism, a disorder of pigmentation in the skin,hair, and eyes. The phenotype of patients with OCA2 is typicallysomewhat less severe than in those with tyrosinase- deficient OCA1.There are several forms of OCA2, from typical OCA to relatively mild‘autosomal recessive ocular albinism’ (AROA). OCA2 is the most prevalenttype of albinism throughout the world. The gene OCA2 is localized tochromosome 15 at 15q11.2- q12 OPN3 opsin 3 eye May play a role inencephalic photoreception. Strongly development expressed in brain.Highly expressed in the preoptic area and paraventricular nucleus of thehypothalamus. Shows highly patterned expression in other regions of thebrain, being enriched in selected regions of the cerebral cortex,cerebellar Purkinje cells, a subset of striatal neurons, selectedthalamic nuclei, and a subset of interneurons in the ventral horn of thespinal cord. OPN5 opsin 5 eye Associated with visual perception andphototransduction. development OTX2 orthodentiele retinal Probably playsa role in the development of the brain and homolog 2 development thesense organs. Defects in OTX2 are the cause of (Drosophila) syndromicmicrophthalmia 5 (MCOPS5). Microphthalmia is a clinically heterogeneousdisorder of eye formation, ranging from small size of a single eye tocomplete bilateral absence of ocular tissues. Up to 80% of cases ofmicrophthalia occur in association with syndromes that includenon-ocular abnormalities such as cardiac defects, facial clefts,microcephaly and hydrocephaly. MCOPS5 patients manifest unilateral orbilateral microphthalmialelinical anophthalmia and variable additionalfeatures including coloboma, microcornea, cataract, retinal dystrophy,hypoplasia or agenesis of the optic nerve, agenesis of the corpuscallosuln, developmental delay, joint laxity, hypotonia, and seizures.PAX6 paired box RPE Transcription factor with important functions in thegene 6 development development of the eye, nose, central nervous systemand (aniridia, pancreas. Required for the differentiation of pancreatickeratitis) islet alpha cells (By similarity). Competes with PAX4 inbinding to a common element in the glucagon, insulin and somatostatinpromoters (By similarity), Isoform 5a appears to function as a molecularswitch that specifies target genes. Defects in Pax6 results in a numberof eye defects and malformations. PHC2 polyhomeotic- development/Component of the Polycornb group (PcG) multiprotein like 2differentiation PRC1 complex, a complex required to maintain the(Drosophila) transcriptionally repressive state of many genes, includingHox genes, throughout development, PcG PRC1 complex acts via chromatinremodeling and modification of histones; it mediates monoubiquitinationof histone H2A ‘Lys-119’, rendering chromatin heritably changed in itsexpressibility. PKNOX2 PBX/knotted 1 developmem/ Known to be involved indevelopment and may, along homeobox 2 differentiation with MEIS, controlPax6. PRKCA protein kinase cellular Very important for cellularsignaling pathways such as C, alpha signalling the MAPK, Wnt, PI3, VEGFand Calcium pathways. PROX1 prospero- eye May play a fundamental role inearly development of related development CNS. May regulate geneexpression and development of homeobox 1 postmitotic undifferentiatedyoung neurons. Highly expressed in lens, retina, and pancreas. PRRX1paired related development/ Necessary for development. Transcriptionalcoactivator, homeobox 1 differentiation enhancing the DNA-bindingactivity of serum response factor. RAI1 retinoic acid development/ Mayfunction as a transcriptional regulator. Regulates induced 1differentiation transcription through chromatin remodeling byinteracting with other proteins in chromatin as well as proteins in thebasic transcriptional machinery. May be important for embryonic andpostnatal development. May be involved in neuronal differentiation. RARAretinoic acid development/ This is a receptor for retinoic acid. Thismetabolite has receptor, alpha differentiation profound effects onvertebrate development. This receptor controls cell function by directlyregulating gene expression. RARB retinoic acid development/ This is areceptor for retinoic acid. This metabolite has receptor, betadifferentiation profound effects on vertebrate development. Thisreceptor controls cell function by directly regulating gene expression.RARRES1 retinoic acid development/ Associated with differentiation andcontrol receptor differentiation proliferation. May be a growthregulator that mediates responder some of the growth suppressive effectsof (tazarotene retinoids. induced) 1 RAX retina and eye Plays a criticalrole in eye formation by regulating the anterior neural developmentinitial specification of retinal cells and/or their fold homeoboxsubsequent proliferation. Binds to the photoreceptor conserved element-I(PCE-1/Ret 1) in the photoreceptor cell-specific arrestin promoter. RB1retinoblastomal development/ An important regulator of other genes andcell growth, (including differentiation Defects in RB1 are the cause ofchildhood cancer osteosarcoma) retinoblastoma (RB). RB is a congenitalmalignant tumor that arises from the nuclear layers of the retina. RDH5retinol RPE retinol dehydrogenase 5,11-cis,expressed in retinaldehydrogenase development pigmented epithelium,formerly RDH1.Stereospecific 5 (11-cis/9-cis) 11-cis retinol dehydrogenase, whichcatalyzes the final step in the biosynthesis of 11-cis retinaldehyde,the universal chromophore of visual pigments. Abundant in the retinalpigmented epithelium. Defects in RDH5 are a cause of fundusalbipurictatus (FA). FA is a rare form of stationary night blindnesscharacterized by a delay in the regeneration of cone and rodphotopigments. RGR retinal G RPE Preferentially expressed at high levelsin the retinal protein development pigmented epithelium (RPE) andMueller cells of the coupled neural retina. Retinal opsin related,(rhodopsin receptor homolog)expressed in the retinal pigmentedepithelium, encoding a retinaldehyde, preferentially all-trans retinal,binding protein, G protein coupled receptor superfamily. RLBP1/CRALretinaldehyde RPE Carries 11-cis-retinol and 11-cis-retinaldehyde as BP1binding development endogenous ligands and may be a functional componentprotein 1 of the visual cycle. Defects in RLBP1 are a cause of autosomalrecessive retinitis pigmentosa (arRP). Retinitis pigmentosa (RP) leadsto degeneration of retinal photoreceptor cells. Defects in RLBP1 are thecause of Bothnia retinal dystrophy, also known as Vasterbottendystrophy. It is another form of autosomal recessive retinitispigmentosa. Defects in RLBP1 are the cause of Newfoundland rod- conedystrophy (NFRCD). NFRCD is a retinal dystrophy reminiscent of retinitispunctata albescens but with a substantially lower age at onset andmore-rapid and distinctive progression. RPE65 retinal pigment RPERetinal pigmented epithelium specific. Retinal epithelium- developmentpigmented epithelium-specific 65, major microsomal specific protein,minor role in the isomerisation of ail-trans to 11- protein 65 kDa cisretinal, associated with the endoplasmic reticulum, also expressed inrenal tumor cells. Plays important roles in the production of 11-cisretinal and in visual pigment regeneration. RRH retinal pigment RPEFound only in the eye, where it is localized to the retinal epithelium-development pigment epithelium (RPE). In the RPE, it is localized toderived the microvilli that surround the photoreceptor outer rhodopsinsegments. May play a role in rpe physiology either by homolog detectinglight directly or by monitoring the concentration of retinoids or otherphotoreceptor-derived compounds. RTN1 reticulon 1 development/ Expressedin neural and neuroendocrine tissues and cell differentiation culturesderived therefrom. Expression of isoform RTN1- C is strongly correlatedwith neuronal differentiation. RXRB retinoid X development/ Nuclearhormone receptor. Involved in the retinoic acid receptor, betadifferentiation response pathway. Binds 9-cis retinoic acid (9C-RA),obligate member of heterodimeric nuclear receptors,steroid/thyroid/retinoic receptor superfamily. RXRG retinoid Xdevelopment/ Nuclear hormone receptor. Involved in the retinoic acidreceptor, differentiation response pathway. Binds 9-cis retinoic acid(9C-RA), gamma obligate member of heterodimeric nuclear receptors,steroid/thyroid/retinoic receptor superfamily. SERPINF1/PE serpin RPESpecific expression in retinal pigment epithelial cells and DF peptidasedevelopment blood plasma. Neurotrophic protein; induces extensiveinhibitor, clade neuronal differentiation in retinoblastoma cells. F(alpha-2 antiplasmin, pigment epithelium derived factor), member 1 SIX3sine oculis eye Expressed during eye development in midline forebrainhomeobox development and in anterior region of the neural plateespecially inner homolog 3 retina and later in ganglion cells and incells of the inner (Drosophila) nuclear layer, involved in regulation ofeye development. SOX10 SKY (sex development/ Transcription factor thatseems to function synergistically determining differentiation with otherdevelopment associated proteins. Could region Y)-box confer cellspecificity to the function of other 10 transcription factors indeveloping and mature glia. SOX5 SRY (sex development/ Expression isassociated with craniofacial, skeletal and determining differentiationcartilage development and is highly expressed in brain, region Y)-boxtestis and various tissues. 5 SOX6 SRY (sex development/ Expression isassociated with craniofacial, skeletal and determining differentiationcartilage development and is highly expressed in brain, region Y)-boxtestis and various tissues. 6 SOX8 SRY (sex development/ May play a rolein central nervous system, limb and determining differentiation facialdevelopment. region Y)-box 8 SOX9 SRY (sex development/ Plays animportant role in the normal development. May determiningdifferentiation regulate the expression of other genes involved forregion Y)-box skeletal and cartilage formation by acting as a 9(campomelic transcription factor for these genes. dysplasia, autosomalsex - reversal) TIMP3 TIMP RPE Matrix metalloprotemase, tissue inhibitor3, expressed in metallopeptidase development retinal pigment epithelium,placenta, localized in inhibitor 3 extracellular matrix. Complexes withmetalloproteinases (Sorsby fundus (such as collagenases) andirreversibly inactivates them. dystrophy, May form part of a tissue-specific acute response to pseudoinflam remodeling stimuli. Defects OnTIMP3 are the cause of . matory) Sorsby fundus dystrophy (SFD). SFD is arare autosomal dominant macular disorder with an age of onset in thefourth decade. It is characterized by loss of central vision fromsubretinal neovascularization and atrophy of the ocular tissues. TTRtransthyretin (prealbumin, Thyroid hormone-binding protein. Probablytransports amyloidosis type I) thyroxine from the bloodstream to thebrain. Defects in TTR are the cause of arnyloidosis VII; also known asleptomeningeal amyloidosis or meningocerebrovascular amyloidosis.Leptomeningeal amyloidosis is distinct from other forms of transthyretinamyloidosis in that it exhibits primary involvement of the centralnervous system. Neuropathologic examination shows amyloid in the wallsof leptomeningeal vessels, in pia arachnoid, and subpial deposits. Somepatients also develop vitreous ainyloid deposition that leads to visualimpairment (oculoleptomeningeal amyloidosis). TYR tyrosinase pigmentedThis is a copper-containing oxidase that functions in the (oculocutancocells formation of pigments such as melanins and other us albinismpolyphenolic compounds. Defects in TYR are the cause IA) ofoculocutaneous albinism type IA (OCA-IA). OCA-I, also known astyrosinase negative oculocutaneous albinism, is an autosomal recessivedisorder characterized by absence of pigment in hair, skin and eyes.OCA-I is divided into 2 types: type IA, characterized by complete lackof tyrosinase activity due to production of an active enzyme, and typeIB characterized by reduced activity of tyrosinase. OCA-IA patientspresents with the life-long absence of melanin pigment after birth andmanifest increased sensitivity to ultraviolet radiation and topredisposition to skin cancer defects in TYR are the cause ofocutocutaneous albinism type IB (OCA-1B); also known as albinism yellowmutant type. OCA-IB patients have white hair at birth that rapidly turnsyellow or blond. TYRP1 tyrosinase- pigmented Specific expression inPigment cells. Oxidation of 5,6- related protein 1 cellsdihydroxyindole-2-carboxylic acid (DHICA) into indole-5,6-quinone-2-carboxylic acid. May regulate or influence the type ofmelanin synthesized. Defects in TYRP1 are the cause of rufousoculocutaneous albinism (ROCA). OCA occurs in blacks and ischaracterized by bright copper-red coloration of the skin and hair anddilution of the color of the iris. Defects-in TYRP1 are the cause ofoculocutaneous albinism type III (OCA-III) also known as OCA3. OCA-IIIis a form of albinism with only moderate reduction of pigment.Individuals with OCA-III are recognized by their reddish skin and haircolor.

TABLE 3 Down regulated genes of interest reported on microarrays GeneSymbol Gene Name Associated with Description ALPL alkaline ES cellsElevated expression of this enzyme is associated with phosphataseundifferentiated pluripotent stem cell. CECR2 cat eye Part of the CERF(CECR2-containing-remodeling syndrome factor) complex, which facilitatesthe perturbation of chromosome chromatin structure in an ATP-dependentmanner. May region, be involved through its interaction with LRPPRC inthe candidate 2 integration of cytoskeletal network with vesiculartrafficking, nucleocytosolic shuttling, transcription, chromosomeremodeling and cytokinesis. Developmental disorders are associated withthe duplication of the gene. DCAMKL1 doublecortin Embryonic Probablekinase that may be involved in a calcium- and CaM development signalingpathway controlling neuronal migration in kinase-like 1 the developingbrain. DPPA2 developmental ES cells May play a role in maintaining cellpluripotentiality,. pluripotency associated 2 DPPA3 developmental EScells May play a role in maintaining cell pluripotentiality.pluripoteney associated 3 DPPA4 developmental ES cells May indicate cellpluripotenliality. pluripotency associated 4 DPPA5/Esg1 developmental EScells Embryonic stem cell marker. pluripotency associated 5/Embryonicstem cellspecific gene 1 FOXD3 fork head box Pluripotence Required formaintenance of pluripotent cells in the D3 pre-implantation andperi-implantation stages of embryogenesis. LIDIECAT11 LINE-1 type EScells Embryonic stem cell marker. transposase domain containing 1/EScell associated transcript 11 NANOG Nanog ES cells Embryonic stem cellmarker. Transcription regulator homeobox involved in inner cell mass andembryonic stem (ES) cells proliferation and self-renewal. Imposespluripotency on ES cells and prevents their differentiation towardsextraembryonic endoderm and trophectoderin lineages. NCAM1 neural cellneuroprogenitors This protein is a cell adhesion molecule involved inadhesion neuron-neuron adhesion, neurite fasciculation, molecule 1outgrowth of neurites, etc. NES/Nestin nestin ES cells Neuralprogenitorcells. NODAL nodal Embryonic Essential for mesoderm formation and axialpatterning development during embryonic development. NR5A2/FTF nuclearEmbryonic May contribute to the development and regulation of receptordevelopment liver and pancreas-specific genes and play importantsubfamily 5, roles in embryonic development. group A, member 2POU5F1/Oct- POU domain, ES cells Embryonic stem cell marker. Indicatorof “Stemness”. 3/4 class 5, Transcription factor that binds to theoctamer motif(5′- transcription ATTTGCAT-3′). Prime candidate for anearly factor 1 developmental control gene. SOX17 SRY (sex Inhibitor ofNegative regulator of the Wnt signalling pathway. determiningdifferentiation region Y)-box 17 SOX2 SRY (sex ES cells Indicator of“Stemness”. Expressed in inner cell mass, determining primitive ectodermand developing CNS. region Y)-box 2 TBX3 T-box 3 (ulnar EmbryonicTranscriptional repressor involved in developmental mammary developmentprocesses. Murine T-box gene Tbx3 syndrome) (T, brachyury) homolog,putative transcription factor, pairing with TBX5, homolog to Drosophilaoptomotor-blind gene (omb), involved in optic lobe and wingdevelopment,involved in developmental regulation, expressed in anteriorand posterior mouse limb buds, widely expressed in adults TDGF1/Criptoteratocarcinom ES cells Indicator of “Stemness”. Could play a role inthe -1 a-derived determination of the epiblastic cells that subsequentlygrowth factor give rise to the mesoderm. 1 TEK/VMCM TEK tyrosine EarlyThis protein is a protein tyrosine-kinase kinase. Endothelialtransmembrane receptor for angiopoietin 1. It may endothelialprogenitors constitute the earliest mammalian endothelial cell (venouslineage marker. Probably regulates endothelial cell malformations,proliferation, differentiation and guides the proper multiple patterningof endothelial cells during blood vessel cutaneous and formationmucosal) TUBB2A, tubulin, beta neuroprogenitors Tubulin is the majorconstituent of microtubules. It TUBB2B 2A, tubulin, binds two moles ofGTP, one at an exchangeable site beta 2B on the beta chain and one at anon-exchangeable site on the alpha-chain. Often associated with theformation of gap junctions in neural cells. TUBB2A, tubulin, betaneuroprogenitors Tubulin is the major constituent of mierotubales. ItTUBB2B, 2A, tubulin, binds two moles of GTP, one at an exchangeable siteTUBB2C, beta 2B, on the beta chain and one at a non-exchangeable site onTUBB3, tubulin, beta the alpha-chain. Often associated with theformation of TUBB4 2C, tubulin, gap junctions in neural cells. beta 3,tubulin, beta 4 TUBB3 tubulin, beta 3 neuroprogenitors Tubulin is themajor constituent of microtubules. It binds two moles of GTP, one at anexchangeable site on the beta chain and one at a non-exchangeable siteon the alpha-chain. Often associated with the formation of gap junctionsin neural cells. TWIST1 twist homolog Inhibitor of Probabletranscription factor, which negatively 1 differentiation regulatescellular determination and differentiation. UTF1 undifferentiated EScells Embryonic stem cell marker. Acts as a transcriptional embryoniccoactivator of ATF2. cell transcription factor 1 VSNL1 visinin-like 1Inhibior of Regulates the inhibition of rhodopsin phosphorylation.rhodopsin ZFP42/Rex-1 zinc finger ES cells Embryonic Stem cell marker.protein 42

The present disclosure demonstrates that human RPE cells can be reliablydifferentiated and expanded from human ES cells under well-defined andreproducible conditions—representing an inexhaustible source of cellsfor patients with retinal degenerative disorders. The concentration ofthese cells would not be limited by availability, but rather could betitrated to the precise clinical requirements of the individual.Repeated infusion or transplantation of the same cell population overthe lifetime of the patient would also be possible if deemed necessaryby the physician. Furthermore, the ability to create banks of matchingor reduced-complexity HLA hES lines from which RPE cells could beproduced could potentially reduce or eliminate the need forimmunosuppressive drugs and/or immunomodulatory protocols altogether.

This disclosure also demonstrates that RPE cells differentiated by themethods described herein express multiple genes that are not expressedby hES cells, fetal RPE cells, or ARPE-19 cells. The unique molecularfingerprint of mRNA and protein expression in the ES-cell derived RPEcells of the invention constitutes a set of markers, such as RPE-65,Bestrophin, PEDF, CRABLP, Otx2, Mit-F, PAX6 and PAX2, that make theseRPE cells distinct from cells in the art, such as hES cells, ARPE-19cells, and fetal RPE cells.

Example 6: Rescue of Visual Function Using RPE Cells from Embryonic StemCells

Certain retinal diseases are characterized by degeneration of theretinal pigment epithelium (RPE) which in turn results in photoreceptorloss. Examples include Stargardt's macular dystrophy in humans and thegenetically-determined dystrophy in the Royal College of Surgeons (RCS)rat. Such a process may also play a role in macular degeneration,affecting more than 10 million people in the US alone.

We investigated conditions under which highly characterized human RPEcells derived from embryonic stem cell lines and manufactured underGMP-compliant conditions could optimally rescue visual function in theRCS rat. MAO1- and MAO9-derived RPE cells were injected into thesubretinal space of 23 day-old (P23) RCS rats, maintainedpost-operatively on oral cyclosporine A immunosuppression. Functionalefficacy was tested by threshold optomotor acuity and luminancethresholds recorded from the superior colliculus. All treated eyes werecompared with sham-injected and untreated eyes. Histological examinationwas performed after these functional assessments.

Experimental results showed a clear dose-response in RCS rats.Administration of a preparation comprising 5×10⁴ RPE cells gave onlyslightly better optomotor thresholds than shams, whereas a preparationcomprising 2×10⁵ RPE cells gave improved performance versus controls.Preparations comprising 5×10⁵ RPE cells produced superior performancethat was sustained over time. Animals performed at 0.48 c/d at P60,significantly (p<0.001) better than shams (0.26 c/d) with some treatedeyes showing normal thresholds (0.6 c/d) and over 0.5 c/d in the bestcases at P90 (sham and untreated animals gave a figure 0.16 c/d, a levelthat indicated substantial visual impairment).

Superior colliculus recordings at P94 also showed much lower luminancethreshold responses in RPE cell-injected eyes with some individualrecordings within the normal range. Histological studies showed donorcells disposed as a semi-continuous, pigmented cell layer immediatelyinternal to endogenous, host RPE. The donor RPE cells were positive forRPE65 and bestrophin, indicating that the transplanted cells were RPEcells and that the cell maintain their cell fate followingtransplantation.

Additionally, transplanted animals maintained photoreceptor thickness incomparison to control animals. The photoreceptors in RPE treatmentanimals were 4-5 cells thick in the rescued area compared with only asingle layer in sham and untreated controls.

The results indicate that well-characterized RPE cells derived fromembryonic stem cells and manufactured under GMP-compliant conditionssurvive after transplantation to the subretinal space of RCS rats, donot migrate into the retina and continue to express moleculescharacteristic of RPE. Most importantly, they achieve significant rescueof visual function in a dose dependent fashion in an animal model ofphotoreceptor degeneration. The data further suggest that these cellsmay be effective in limiting and/or reversing the deterioration ofvision that accompanies RPE-driven photoreceptor degeneration in humandisease.

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All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1-134. (canceled)
 135. A pharmaceutical preparation comprising human RPEcells derived in vitro from human pluripotent stem cells, wherein atleast 95% of cells in the preparation are human RPE cells that expressthree or more of the following markers: RPE-65, Bestrophin, PEDF,CRALBP, Otx2, and Mit-F.
 136. The pharmaceutical preparation of claim135, formulated for administration to a subretinal space of a human eye.137. The pharmaceutical preparation of claim 135, formulated as asuspension, matrix, or substrate.
 138. The pharmaceutical preparation ofclaim 135, wherein the pharmaceutical preparation comprises at least 10⁴human RPE cells, or at least 10⁵ human RPE cells, or at least 10⁶ humanRPE cells.
 139. The pharmaceutical preparation of claim 135, wherein thehuman RPE cells are derived in vitro from human embryonic stem cells.140. The pharmaceutical preparation of claim 135, wherein the human RPEcells are derived in vitro from human induced pluripotent stem cells.141. The pharmaceutical preparation of claim 135, wherein at least 95%of the cells in the preparation are human RPE cells that express 4, 5 or6 of the markers.
 142. The pharmaceutical preparation of claim 135,wherein 99% of the cells in the preparation are human RPE cells thatexpress three or more of the markers.
 143. A method of treating asubject having a condition characterized by retinal degeneration,comprising administering to a subject in need thereof an effectiveamount of a preparation of human RPE cells, wherein the human RPE cellsare derived in vitro from human pluripotent stem cells, and wherein atleast 95% of the cells in the preparation are human RPE cells thatexpress three or more of the following markers: RPE-65, Bestrophin,PEDF, CRALBP, Otx2, and Mit-F.
 144. The method of claim 143, wherein thecondition characterized by retinal degeneration is selected from thegroup consisting of: Stargardt's macular dystrophy, age related maculardegeneration, and retinitis pigmentosa, and optionally wherein thepreparation is (a) formulated as a suspension, matrix, or substrate,and/or (b) administered by injection into a subretinal space of an eyeof the subject.
 145. The method of claim 143, wherein the effectiveamount of the preparation comprises about 10⁴ to about 10⁶ human RPEcells.
 146. The method of claim 143, wherein the human RPE cells arederived in vitro from human embryonic stem cells.
 147. The method ofclaim 143, wherein the human RPE cells are derived in vitro from humaninduced pluripotent stem cells.
 148. The method of claim 143, wherein atleast 95% of the cells in the preparation are human RPE cells thatexpress 4, 5 or 6 of the markers.
 149. The method of claim 143, wherein99% of the cells in the preparation are human RPE cells that expressthree or more of the markers.
 150. The method of claim 143, whereintreatment is determined by measuring electroretinogram responses,optomotor acuity threshold, or luminance threshold in the subject. 151.The method of claim 143, wherein the retinal degeneration is associatedwith photoreceptor loss.
 152. The method of claim 143, wherein the humansubject maintains photoreceptor thickness following administration ofthe preparation of human RPE cells.
 153. The method of claim 152,wherein the photoreceptor thickness is about 4-5 cells thick.
 154. Themethod of claim 143, wherein the subject has viable endogenous host RPEcells prior to administration of the preparation of human RPE cells.